Methods and compositions for ovarian organoid culture

Abstract

In some aspects, methods are provided for producing primordial follicles from human and other mammalian germ cells with high efficiency, high cell quality, and high reproducibility. In some aspects, methods and culture systems are provided for producing ovarian organoids, such as cell aggregates composed of both somatic and germ cells from primary and / or stem cell-derived sources, the ovarian organoids having the ability to advance primordial germ cells into oocytes that are competent to enter meiosis and form follicular aggregates. In some aspects, the embodiments provided include the step of culturing the ovarian organoids in fully normalized serum-free medium and under conditions using a generally low normalized serum protein component.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 497,191, filed on April 19, 2023, entitled "METHODS AND COMPOSITIONS FOR OVARIAN ORGANOID CULTURE," the contents of which are incorporated in their entirety by reference.

[0002] field This disclosure relates generally to methods and compositions for producing ovarian organoids having defined cell types and morphological characteristics. [Background technology]

[0003] background For example, the in vitro induction of gonadal cell populations and organoids from stem cells has the potential to provide powerful tools for studying biology and offering therapies and treatments for conditions related to these reproductive tissues. However, the ability to induce and differentiate various gonadal cell types in culture remains limited. There is a need for novel and improved methods to overcome these challenges. This disclosure addresses these and other needs. [Overview of the Initiative]

[0004] overview In some aspects, a method for generating primordial follicles is provided herein, comprising the steps of (a) providing a cell mixture of germ cells and somatic cells under culture, and (b) culturing the cell mixture in a specified medium for a certain period of time to generate primordial follicles, wherein the specified medium contains a protein or protein substitute, and the concentration of the total protein or protein substitute in the specified medium is less than 3.5 milligrams (mg / mL) per milliliter. In some of the embodiments provided, each primordial follicle comprises (i) an oocyte and (ii) a plurality of granulosa cells. In some aspects, a method for generating oogonia and / or oocytes is provided herein, comprising the steps of (a) providing a cell mixture of germ cells and somatic cells under culture, and (b) culturing the cell mixture in a specified medium for a certain period of time to generate oogonia and / or oocytes, wherein the specified medium contains a protein or protein substitute, and the concentration of the total protein or protein substitute in the specified medium is less than 3.5 milligrams (mg / mL) per milliliter.

[0005] In some of the embodiments provided, the method includes a step of further culturing oogonia and / or oocytes to generate primordial follicles. In some of the embodiments provided, the further culturing step is carried out in the same or a different medium as the specified medium.

[0006] In some of the embodiments provided, the prescribed medium contains protein or protein substitute, and the concentration of total protein or protein substitute in the prescribed medium is less than 3.5 mg / mL, less than 3.0 mg / mL, less than 2.5 mg / mL, less than 2.0 mg / mL, less than 1.5 mg / mL, less than 1.0 mg / mL, less than 0.5 mg / mL, or less than 0.1 mg / mL. In some of the embodiments provided, the concentration of total protein or protein substitute in the prescribed medium is about 0.1 mg / mL to 0.5 mg / mL, about 0.5 mg / mL to 1.0 mg / mL, about 1.0 mg / mL to 1.5 mg / mL, about 1.5 mg / mL to 2.0 mg / mL, about 2.0 mg / mL to 2.5 mg / mL, about 2.5 mg / mL to 3.0 mg / mL, or about 3.0 mg / mL to 3.5 mg / mL. In some of the embodiments provided, the concentration of total protein or protein substitute in the standard medium is approximately 0.1 mg / mL to 0.5 mg / mL. In some of the embodiments provided, the concentration of total protein or protein substitute in the standard medium is approximately 0.5 mg / mL to 1.0 mg / mL. In some of the embodiments provided, the concentration of total protein or protein substitute in the standard medium is approximately 1.0 mg / mL to 1.5 mg / mL. In some of the embodiments provided, the concentration of total protein or protein substitute in the standard medium is approximately 1.5 mg / mL to 2.0 mg / mL. In some of the embodiments provided, the concentration of total protein or protein substitute in the standard medium is approximately 2.0 mg / mL to 2.5 mg / mL. In some of the embodiments provided, the concentration of total protein or protein substitute in the standard medium is approximately 2.5 mg / mL to 3.0 mg / mL. In some of the embodiments provided, the concentration of total protein or protein substitute in the prescribed medium is approximately 3.0 mg / mL to 3.5 mg / mL.In some of the embodiments provided, the concentration of total protein or protein substitute in the specified culture medium is 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, or 3.5 mg / mL, or approximately 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, or 3.5 mg / mL, or any of the values ​​mentioned above.

[0007] In some of the embodiments provided, the total concentration of protein or protein substitute in the standard medium is less than or equal to the concentration of protein in the medium containing 7.5% (v / v) FBS. In some of the embodiments provided, the standard medium does not contain serum. In some of the embodiments provided, the standard medium does not contain FBS.

[0008] In some of the embodiments provided, the standard medium comprises one or more components selected from inorganic salts, sugars, amino acids, vitamins, organic acids, antioxidants, and buffers; as well as proteins or protein substitutes. In some of the embodiments provided, the standard medium comprises proteins or protein substitutes in the basal medium. In some of the embodiments provided, the basal medium comprises Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle Basal Medium (BME), F-10, F-12, RPMI 1640, Glasgow Minimum Essential Medium (GMEM), Alpha Minimum Essential Medium (AlphaMEM), Advanced Minimum Essential Medium (AdvancedMEM), Iskov Modified Dulbecco Medium, or M199.

[0009] In some of the embodiments provided, the protein or protein substitute in the culture medium is a protein. In some of the embodiments provided, the protein or protein substitute in the culture medium is a protein substitute. In some of the embodiments provided, the protein includes serum protein components. In some of the embodiments provided, the protein includes mammalian serum protein components. In some of the embodiments provided, the serum protein components are defined. In some of the embodiments provided, the protein or protein substitute is albumin or an albumin substitute, or includes it. In some embodiments, the protein is albumin, or includes it. In some embodiments, the protein substitute is an albumin substitute, or includes it. In certain examples, if the protein is albumin, the protein substitute is an albumin substitute. In some of the embodiments provided, the protein or protein substitute comprises one or more components selected from bovine pituitary gland extract, plant hydrolysates (e.g., rice hydrolysates), albumin, chick extract, bovine embryo extract, bovine fetal albumin (fetuin), egg albumin, human serum albumin (HSA), albumin of other animal origin, or bovine serum albumin. In some of the embodiments provided, the protein or protein substitute is or comprises lipid-enriched albumin. In some of the embodiments provided, the protein or protein substitute is or comprises lipid-enriched bovine serum albumin. In some of the embodiments provided, the protein or protein substitute is or comprises AlbumX® lipid-rich bovine serum albumin, optionally AlbumX® I lipid-rich bovine serum albumin, or AlbumX® II lipid-rich bovine serum albumin. In some of the embodiments provided, the protein or protein substitute comprises synthetic polymers.In some of the embodiments provided, the synthetic polymer is polyvinyl alcohol (PVA) and / or polyvinylpyrrolidone (PVP). In some embodiments, protein substitute refers to any compound that can substitute for a protein in a culture medium for use in the methods herein to give results substantially similar to those of a protein. In some embodiments, the protein or protein substitute in the prescribed medium aids in cell adhesion, growth, proliferation, and maintenance. In some of the embodiments provided, the prescribed medium is prepared by adding the protein or protein substitute to a basic medium. In some of the embodiments provided, the protein or protein substitute is provided by a supplement medium that is added to the basic medium. In some of the embodiments provided, the supplement medium is a prescribed serum substitute medium. In some of the embodiments provided, the supplement medium is added to the basic medium to a final concentration of 7.5% (v / v) or less. In some of the embodiments provided, the supplement medium is added to the base medium to a final concentration of approximately 1%–7.5%, 1%–5%, 1%–3%, 3%–5%, or 5%–7.5% (all v / v). In some of the embodiments provided, the supplement medium is added to the base medium to a final concentration of 0.5% or about 0.5%, 1.0% or about 1.0%, 1.5% or about 1.5%, 2.0% or about 2.0%, 2.5% or about 2.5%, 3.0% or about 3.0%, 3.5% or about 3.5%, 4.0% or about 4.0%, 4.5% or about 4.5%, 5.0% or about 5.0%, 5.5% or about 5.5%, 6.0% or about 6.0%, 6.5% or about 6.5%, 7.0% or about 7.0%, or 7.5% or about 7.5%, or any of the values ​​above (all percentages are v / v). In some of the embodiments provided, the supplement medium is added to the base medium to a final concentration of 2%.In some of the embodiments provided, the supplement medium comprises one or more components selected from the group consisting of albumin or an albumin substitute, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some of the embodiments provided, the supplement medium comprises albumin or an albumin substitute. In some of the embodiments provided, the supplement medium comprises an antioxidant selected from the group consisting of reduced glutathione, ascorbic acid, and ascorbic acid-2-phosphate. In some of the embodiments provided, the supplement medium comprises a collagen precursor selected from the group consisting of L-proline and its polymers or derivatives, L-hydroxyproline and its polymers or derivatives, and ascorbic acid or its polymers. In some of the embodiments provided, the supplement medium includes a transferrin substitute which is an iron chelate, such as ferric citrate chelate and ferrous sulfate chelate, and optionally an iron chelate selected from the group consisting of ferrous sulfate-7-hydrate-EDTA. In some of the embodiments provided, the supplement medium includes an insulin substitute selected from the group consisting of zinc chloride, zinc bromide, and zinc sulfate-7-hydrate. In some of the embodiments provided, the supplement medium includes an amino acid component which comprises one or more amino acids selected from the group consisting of glycine, L-alanine, L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-glutamine, L-arginine, L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, and their derivatives. In some of the embodiments provided, the supplement medium is Ag. + , Al3 + , Ba2+ , Cd 2+ , Co 2+ , Cr 3+ , Ge 4+ , Se 4+ , Br - , I - , Mn 2+ , F - , Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn 2+ , and Zr 4+ It contains a trace element component containing one or more trace element parts selected from the group consisting of. In some of any of the provided embodiments, the supplement medium is lipid-rich albumin (AlbuMAX), L-glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, sodium selenite, Ag + , Al3[[ID=3​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​In some of any of the provided embodiments, the supplement medium is Knockout (TM) Serum Replacement (KSR). In some of any of the provided embodiments, the defined medium is a basal medium supplemented with 1-5% of a defined Knockout (TM) Serum Replacement (KSR). In some of any of the provided embodiments, the defined medium is a basal medium supplemented with approximately 2% of a defined Knockout (TM) Serum Replacement (KSR).

[0011] In some of any of the provided embodiments, the cell mixture is or forms ovarian organoids. In some of any of the provided embodiments, the ovarian organoids are cultured in a complete immersion suspension culture. In some of any of the provided embodiments, the ovarian organoids are cultured at an air-liquid interface using a permeable culture membrane. In some of any of the provided embodiments, the method further comprises removing the defined medium from the cell mixture after oogonia and / or oocytes are generated. In some of any of the provided embodiments, the ovarian organoids are composed of 500 to 300,000 cells. In some of any of the provided embodiments, the percentage of germ cells in the ovarian organoids is 1% to 20% or 1% to 50% of the total number of cells in the organoids.

[0012] In some of any of the provided embodiments, the cell mixture is cultured in vitro for 1 day to 300 days.

[0013] In some of the embodiments provided, the method further includes a step of activating a primordial follicle to generate a primary follicle. In some of the embodiments provided, the germ cells are primordial germ cells (PGCs). In some of the embodiments provided, the germ cells are primordial germ cell-like cells (PGCLCs). In some of the embodiments provided, the germ cells express one or more genes selected from TFAP2C, PRDM1, and POU5F1. In some of the embodiments provided, the germ cells are human. In some of the embodiments provided, the germ cells are derived from in vivo tissue. In some of the embodiments provided, the germ cells are derived from pluripotent stem cells (PSCs). In some embodiments, the PSCs are induced pluripotent stem cells (iPSCs). In some of the embodiments provided, the germ cells differentiate into oogonia and / or oocytes. In some of the embodiments provided, the oogonia express one or more genes selected from DDX4, DAZL, STRA8, SYCP3, and SYCP1. In some of the embodiments provided, the oocyte expresses one or more genes selected from FIGLA and ZP3.

[0014] In some of any of the provided embodiments, the somatic cell is a mammalian ovarian somatic cell. In some of any of the provided embodiments, the somatic cell is collected in vivo. In some of any of the provided embodiments, the somatic cell is collected from an in vivo mammalian fetal ovary. In some of any of the provided embodiments, the somatic cell is derived from a human, non-human primate, pig, rabbit, cow, mouse, rat, donkey, and / or rabbit. In some of any of the provided embodiments, the somatic cell is differentiated in vitro from another cell type. In some of any of the provided embodiments, the somatic cell is derived from pluripotent stem cells (PSCs). In some embodiments, the PSC is an induced pluripotent stem cell (iPSC). In some of any of the provided embodiments, the somatic cell includes one or more ovarian somatic cell types. In some of any of the provided embodiments, one or more of the somatic cell types express one or more genes characteristic of the intermediate mesoderm, including WT1. In some of any of the provided embodiments, one or more of the somatic cell types express one or more genes characteristic of the coelomic epithelium, including WT1 and GATA4. In some of any of the provided embodiments, one or more of the somatic cell types express one or more genes characteristic of granulosa cells, including WT1, GATA4, LHX9, NR5A1, and / or FOXL2. In some of any of the provided embodiments, one or more of the somatic cell types express one or more genes characteristic of bipotential gonads, including WT1, GATA4, LHX9, and / or NR5A1.

[0015] The present invention provides a method for generating an ovarian organoid comprising one or more primordial follicles, wherein each primordial follicle comprises (i) an oocyte and (ii) a plurality of granulosa cells, and the oocyte is derived from a human or non-human primate precursor. In some of the embodiments provided, the oocyte is derived from a human germ cell. In some embodiments, the human germ cell is derived from a stem cell. In some of the embodiments provided, the ovarian organoid comprises at least five primordial follicles, at least ten primordial follicles, at least twenty primordial follicles, at least fifty primordial follicles, at least one hundred primordial follicles, at least five hundred primordial follicles, or more than five hundred primordial follicles. In some of the embodiments provided, the ovarian organoid is derived from a cell mixture of ovarian germ cells and somatic cells under culture, and the ovarian organoid contains at least 5, at least 10, at least 50, at least 100, or at least 500 oocytes and / or oogonia derived from ovarian germ cells, and the ovarian germ cells are of human or non-human primate origin. In some of the embodiments provided, the ovarian germ cells are human germ cells. In some of the embodiments provided, the ovarian germ cells are primary germ cells (PGCs) or primary germ cell-like cells (PGCLCs). In any of the embodiments provided, the percentage of oocytes, oogonia, and / or other germ cells in the ovarian organoid is about 1% or more than 1%, about 5% or more than 5%, about 10% or more than 10%, about 20% or more than 20%, about 30% or more than 30%, about 40% or more than 40%, or about 50% or more than 50% of the total number of cells in the ovarian organoid. [Brief explanation of the drawing]

[0016] [Figure 1]Exemplary results are shown from culturing ovarian organoids in fully normalized medium containing 2% (v / v) normalized serum substitute (DSR) (e.g., 2% KSR), demonstrating a significant improvement in PGC survival at 21 days in organoid cultures compared to culture in 10% DSR, and also compared to standard published methods, i.e., culture in long-term medium containing 2% or 10% (v / v) FBS. Left panel: Histological sections of ovarian organoids cultured in medium containing 2% (v / v) DSR, 2% (v / v) FBS, 10% (v / v) DSR, or 10% (v / v) FBS. White arrows indicate surviving PGCs positively co-stained for the hPSC-derived reporter marker and the PGC marker AP2g. Structures indicated by asterisks are hPSC-derived cells that have lost their PGC identity. Right panel: Quantification of mean viable PGCs in three distinct histological sections at 21 days for each group. [Figure 2] Exemplary images demonstrating the robust survival of PSC-derived germ cells in ovarian organoids cultured in standardized media (long-term media containing a standardized serum substitute (e.g., KSR)). Ovarian organoids were prepared by mixing human iPSC-derived female germ cells from a fluorescently tagged reporter cell line with ovarian somatic cells. Images show time-course bright-field (images of all cells; upper left of each panel) and fluorescence (visualization of fluorescently tagged PSC-derived germ cells; upper right of each panel) separately and superimposed (larger image for each panel) in culture under either standardized media (2% (v / v) DSR) or 10% (v / v) FBS conditions. [Figure 3]Figures 3A–3C show exemplary images demonstrating the generation of PSC-derived female germ cells that have progressed appropriately in terms of eligibility for follicular aggregation. Prior to follicular aggregation within the developing mammalian gonad, female germ cells develop through defined stages of germ cell progression. After meiotic initiation and subsequent meiotic arrest, female germ cells begin to express markers including LMOD3 and ZP3, signaling eligibility for follicular aggregation. Figure 3A shows a histological section of a human fetal ovary at 17 weeks of gestation, where germ cells express LMOD3 in the cytoplasm and ZP3 around the cell periphery. Figure 3B shows a histological section of an ovarian organoid cultured for a long period under defined serum conditions. Many cells in this section express LMOD3 in the cytoplasm and ZP3 around the cell periphery. Nuclear reporters indicate that these cells are PSC-derived. Figure 3C shows an ovarian organoid cultured for a long period under 10% (v / v) FBS serum conditions. In this group, there are few or no cells expressing LMOD3 or ZP3. Overall, far fewer cells are positive for PSC-derived reporters. [Figure 4A] Figures 4A and 4B show exemplary results demonstrating that prescribed serum conditions lead to robust survival, progression, and follicular aggregation of PSC-derived germ cells in ovarian organoids compared to 10% (v / v) FBS conditions. The left and right panels of Figure 4A show histological sections of ovarian organoids generated from human PSC-derived germ cells and ovarian somatic cells cultured for extended periods under prescribed serum conditions (left) or 10% (v / v) FBS conditions (right), stained for the oogonia marker DDX4, the granulosa somatic cell marker FOXL2, and the cytoplasmic reporter for PSC-derived cells. Organoids cultured under prescribed serum conditions contain a rich number of oogonia and granulosa cells beginning to organize into primordial follicles. Organoids cultured under 10% (v / v) FBS conditions contain large areas of completely cell-free tissue, with very low PSC-derived germ cell survival and very few FOXL2+ granulosa cells. Figure 4B shows the same histological sections with fluorescent markers separated into individual channels. [Figure 4B] Please refer to the explanation in Figure 4A. [Figure 5]This paper presents exemplary results demonstrating the efficient induction of primordial follicles from PSC-derived germ cells in in vitro ovarian organoids under specified serum conditions. The upper left panel shows a histological section of a 19-week-old human fetal ovary stained with the oogonia / oocyte marker DDX4 in the cytoplasm of germ cells, with nuclear FOXL2 staining marking surrounding somatic granulosa cells. The two panels on the right show histological sections of ovarian organoids cultured long-term under specified serum conditions, where DDX4 stains oogonia / oocytes in the cytoplasm, and FOXL2 stains the nuclei of surrounding somatic granulosa cells. At the far right, the cytoplasmic reporter defines PSC-derived cells. All PSC-derived cells within the organoid are DDX4+ and surrounded by organized FOXL2+ granulosa cells. The bottom three rows of the panel are selected magnified images of examples of follicular structures found in larger sections of ovarian organoids cultured for extended periods under specified serum conditions: the bottom panel is a cytoplasmic reporter defining PSC-derived cells; the middle panel is nuclear FOXL2 staining; and the top panel is an overlay of DDX4 and FOXL2 staining from the bottom two rows. Cells indicated by asterisks are DDX4+ germ cells. [Figure 6A]Figures 6A–6C show uniform manifold approximation and projection (UMAP) graphs of single-cell RNA sequencing data comparing in vivo control human fetal germ cells at 6–22 weeks post-conception (pwc), in vitro human pluripotent stem cell-derived germ cells from primordial germ cell-like cells (PGCLCs) initiated in cell mixture, and / or in vitro germ cells after culturing ovarian organoids in a defined medium containing a defined serum substitute (e.g., KSR). Figure 6A shows exemplary results indicating that in vivo control human fetal germ cells span developmental stages 1 through 4. Figure 6B shows exemplary results of single-cell RNA sequencing data of in vivo control human fetal germ cells merged with human pluripotent stem cell-derived germ cells from PGCLCs initiated in cell mixture. Figure 6C shows exemplary results of single-cell RNA sequencing data of in vivo control human fetal germ cells merged with germ cells obtained from ovarian organoids. Following culture of ovarian organoids in standard medium, a subset of in vitro cultured human germ cells derived from pluripotent stem cells in the ovarian organoids directly overlapped with human fetal germ cells spanning stages 1 through 4, indicating the progression of in vitro cultured germ cells in this culture system. Single-cell RNA-seq was performed using the 10x Genomics platform. [Figure 6B] Please refer to the explanation in Figure 6A. [Figure 6C] Please refer to the explanation in Figure 6A. [Figure 7] The images show control human fetal ovaries (upper panel) and cultured ovarian organoids (lower panel) stained with germ cell markers (DDX4+) that stain the cell pericellular area (a subset of stains indicated by arrows in the overlay image), and with nuclear SYCP3+ staining (a subset of stains indicated by arrows in the overlay image), an indicator of meiotic transition. [Modes for carrying out the invention]

[0017] Detailed explanation This specification provides in vitro methods for generating follicles, such as primary or primordial follicles, as well as oogonia and / or oocytes. In some of the methods provided, follicles (e.g., primordial follicles) are produced by culturing somatic cells and germ cells in a cell mixture. For example, cells are cultured in a prescribed medium, particularly in a prescribed medium having a low total protein concentration (where germ cells and somatic cells in the cell mixture aggregate to form ovarian organoids), and also in an embodiment (where germ cells in the ovarian organoids further differentiate into oogonia and / or oocytes). In other embodiments, culturing the ovarian organoids formed in a prescribed medium further generates follicles, such as primary or primordial follicles, which contain follicles containing oocytes surrounded and / or in contact with a plurality of granulosa cells, e.g., somatic cells. Thus, the method provides conditions, including the preparation of a culture medium, for supporting in vitro follicular development and the maturation of oogonia and oocytes.

[0018] For example, it is shown herein that culturing a mixture of germ cells and somatic cells in a serum-deficient medium containing low concentrations of protein or protein substitutes generates primordial follicles, oogonia, and oocytes. In the provided embodiment, primordial follicles, oogonia, and oocytes can be generated under culture conditions in which the concentration of total protein or protein substitute in the culture medium is less than 3.5 mg / mL.

[0019] Low percentages or concentrations of proteins or protein substitutes in prescribed media provided herein can provide benefits to the culture of desired cell types compared to higher percentages of protein or serum. For example, the benefits of low percentages of proteins or protein substitutes in prescribed media may include improved germ cell survival, reduced off-target growth from unprescribed media, and improved germ cell progression to induce primordial follicle development. As shown herein, prescribed media with low concentrations or percentages of proteins or protein substitutes can support improved follicle generation, such as primordial follicles, compared to culture conditions using media used in published protocols for ovarian organoid culture, such as media containing FBS (e.g., 10% (v / v) FBS). Follicle generation in prescribed media containing low levels of protein or protein substitutes, such as 3.5 mg / mL or approximately 3.5 mg / mL of total protein or protein substitute, can result in increased follicle generation compared to cultures containing higher levels of protein or protein substitutes or serum in the same medium.

[0020] During mammalian fetal development, germ cells develop in the gonads. The gonads are organs that produce gametes (sperm and eggs) in males and females. In the early stages of fetal development, the gonads are undifferentiated and have the potential to develop into either testes or ovaries.

[0021] The development of germ cells in the fetal gonads begins with the migration of primordial germ cells (PGCs) from the yolk sac to the developing gonad. PGCs are precursor cells of the egg and sperm and arise early in fetal development. PGCs are initially located in the epiblast, the outer layer of the blastocyst. They then migrate to the developing gonad, guided by signals from the surrounding tissues.

[0022] When PGCs reach the gonads, they begin to interact with the surrounding somatic cells, which are the non-germ cells of the gonads. The somatic cells provide signals that instruct the germ cells to differentiate and begin meiosis, which is the process of cell division that produces haploid gametes.

[0023] In females, developing ovarian somatic cells promote the differentiation of germ cells into oogonia, which are cells committed to becoming eggs. During division, oogonia form structures called nests, surrounded by granulosa cells. As development progresses, oogonia begin to differentiate into oocytes, which then halt after meiosis I. At this point, a process called nest collapse begins, in which granulosa cells migrate between oocytes and rearrange to form structures called primordial follicles. These follicles remain dormant until puberty, when some of them begin to reactivate. Once activated, the follicles and the oocytes within them begin to grow in size. When oocytes reach a certain size, they are ready to mature into eggs that can be fertilized and produce an embryo.

[0024] Part of this process can be replicated in vitro, thereby allowing mammalian primordial germ cells to be induced from pluripotent stem cells using various techniques. Multiple protocols exist, differing slightly by species, but primarily utilize BMP signaling to differentiate mammalian pluripotent stem cells (PSCs) into PGCs (Irie et al., 2015; Sasaki et al., 2015; Kobayashi et al., 2021; Sakai et al., 2020; Seika et al., 2020). Some of these methods include intermediate stages, while others involve the overexpression of specific transcription factors or genes of the GATA family (Hamazaki et al., 2020; Kojima et al., 2021; Kramme et al., 2022).

[0025] In mice, PGCs derived in vitro or in vivo can be further developed into oogonia and oocytes with the help of fetal ovarian somatic cells derived in vivo or in vitro sources, ultimately forming follicles (Hikabe et al., 2016; Yoshino et al., 2021). Using a similar model, human and non-human primate PGCs were advanced to oogonia and even to meiosis by co-culturing them with mouse fetal ovarian somatic cells in a high-serum-content culture method. However, this differentiation process remains highly inefficient, with the majority of germ cells dying and only a small number progressing (Yamashiro et al., 2018; Murase et al., 2020; Gyobu-Motani et al., 2023). To date, there has been no previous system that has been able to robustly advance a large number of human cells to the oogonia and meiotic phases, nor has there been any previous system that has been able to advance in vitro-induced human germ cells to the oocyte or follicular phase, which are crucial stages for generating eggs from somatic cells in vitro.

[0026] The methods provided herein overcome previous limitations and can be used to prepare ovarian organoids in vitro. In several aspects, the compositions and methods provided address the lack of human germ cell viability in ovarian organoids in previous culture methods. In several aspects, the methods and compositions provided, including culture with smaller amounts of protein or protein substitutes than conventional methods, induce robust germ cell progression to oocytes capable of forming primordial follicles. In several aspects, the methods and compositions provided induce robust generation of ovarian primordial follicles that closely resemble in vitro follicles in early to late pregnancy and adult ovarian primordial follicles. In several aspects, the methods and compositions provided include the use of a standard medium for long-term culture of ovarian organoids. In several aspects, the standard medium is a fully standard medium containing low overall percentages or concentrations of protein and / or protein substitute components.

[0027] As described herein, low percentages or concentrations of proteins or protein substitutes in the prescribed media provided herein can improve germ cell viability, reduce off-target growth from unprescribed media, and enable germ cell progression, allowing for the rapid and robust generation of primordial follicular development. Culturing germ cells in a serum-free medium containing these low levels of protein, compared to conventional methods, increases cell viability after extended periods of culture (e.g., 21 days). For example, reducing the amount of protein in cell culture media, as shown herein, can increase the viability of PGCLCs and germ cells. The prescribed media used herein are serum-free and contain less protein (e.g., 3.5 mg / mL or approximately 3.5 mg / mL) or protein substitutes compared to conventional methods cultured in the presence of serum containing a mixture of proteins. In some aspects, culturing ovarian organoids in prescribed media significantly improves germ cell viability, proliferation, differentiation, and follicular formation. In some aspects, this improvement is comparable to current standard culture conditions that rely on culture in a medium containing fetal bovine serum.

[0028] The Specified also provides ovarian organoids, such as ovarian organoids produced using any of the methods or compositions provided herein. In some embodiments, the ovarian organoids include germ cells, such as oogonia and / or oocytes, derived from precursors, such as primordial germ cells. In some embodiments, germ cells (e.g., primordial germ cells) are in vitro induced from pluripotent stem cells (PSCs), such as induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the ovarian organoids include ovarian somatic cells that can support the survival, proliferation, and / or differentiation of germ cells. In some embodiments, the ovarian organoids include follicles, such as primordial follicles. In some embodiments, follicles, such as primordial follicles, may be further advanced (e.g., activated).

[0029] Methods for generating primordial follicles in several aspects are provided herein. In some aspects, the method comprises the steps of (a) providing a cell mixture of germ cells and somatic cells under culture, and (b) culturing the cell mixture in a specified medium for a certain period of time to generate primordial follicles. Methods for generating oogonia and / or oocytes in several aspects are provided herein. In some aspects, the method comprises the steps of (a) providing a cell mixture of germ cells and somatic cells under culture, and (b) culturing the cell mixture in a specified medium for a certain period of time to generate oogonia and / or oocytes. In some aspects, the specified medium contains a protein or protein substitute, and the concentration of the total protein or protein substitute in the specified medium is less than 3.5 milligrams (mg / mL) per milliliter.

[0030] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference in whole for all purposes to the same extent as each individual publication is incorporated by reference individually. If any definition contained herein contradicts or otherwise conflicts with any definition contained herein in a patent, application, published application, or other publication incorporated by reference, the definition contained herein shall prevail over the definition incorporated by reference.

[0031] The section headings used herein are for organizational purposes only and should not be construed as limiting the subjects described.

[0032] I. Ovarian organoid culture In some embodiments, methods are provided for culturing gonadal cells, such as ovarian cells, in vitro (e.g., under culture conditions). In some embodiments, the method comprises culturing germ cells and somatic cells together to form a cell mixture. In some embodiments, the cell mixture is cultured in a prescribed medium, such as any prescribed medium provided herein and / or described in Section II and the Examples. In some embodiments, the cell mixture is cultured in a prescribed medium that is serum-free and contains low concentrations or percentages of protein or protein substitutes.

[0033] In some embodiments, the method involves culturing germ cells and somatic cells together in a prescribed medium containing a protein or protein substitute to form a cell mixture, wherein the total concentration of the protein or protein substitute in the prescribed medium is less than 3.5 mg / mL in the absence of serum. In some examples, the cell mixture of germ cells and somatic cells can be further cultured in a prescribed medium containing a total of less than 3.5 mg / mL of protein or protein substitute to form ovarian organoids. In some embodiments, the ovarian organoids are cultured in a prescribed medium containing a low concentration (e.g., less than about 3.5 mg / mL) of total protein or protein substitute until oocytes and / or oogonia are induced. In some embodiments, the ovarian organoids prepared from culturing germ cells and somatic cells in a culture medium containing a small amount of protein or protein substitute are subsequently further cultured until follicles are formed. In some embodiments, after the ovarian organoids have been formed in the low-protein or protein substitute culture, the ovarian organoids can be further cultured in the prescribed medium or in a different medium. In aspects herein, culturing germ cells, somatic cells, and / or ovarian organoids in a prescribed medium as described herein, which contains less protein than a standard culture medium preparation, increases germ cell survival, proliferation, and differentiation, thereby supporting the generation of oocytes, oogonia, and / or follicles (e.g., primordial follicles) in ovarian organoid cultures.

[0034] In several aspects, methods for generating primordial follicles, such as by culturing ovarian cells in vitro, are provided herein. In some aspects, ovarian cells are cultured as ovarian organoids. In some aspects, the ovarian organoids are a mixture of aggregated cell types containing one or more ovarian cell types, such as any of the ovarian cell types described herein, including ovarian germ cells or somatic cells. In some aspects, the ovarian organoids are cultured in a long-term culture, for example, in vitro for 1 to 300 days.

[0035] In some embodiments, the methods described herein provide efficient generation of ovarian germ cells such as oocytes and / or oogonia. In some embodiments, the methods enable the generation of follicles such as primordial follicles. In some embodiments, the primordial follicle comprises an oocyte surrounded and / or in contact with a plurality of granulosa cells. In some embodiments, the primordial follicle can be further activated to generate, for example, a primary follicle.

[0036] In some embodiments, the ovarian organoid is an aggregated mixture of cell types comprising one or more ovarian cell types, such as germ cells and / or somatic cells as described herein, as described in Section IB. In some embodiments, the ovarian organoid culture or ovarian organoid comprises germ cells.

[0037] In some embodiments, ovarian organoids include germ cells and / or somatic cells.

[0038] In some embodiments, germ cells and / or somatic cells are mammalian cells. Germ cells and / or somatic cells in embodiments herein may originate from a variety of sources, including but not limited to humans, pigs, rabbits, cattle, mice, rats, donkeys, rabbits, non-human primates, or other mammalian species. Germ cells and somatic cells do not need to be of the same species. In some embodiments, germ cells are human. In some embodiments, somatic cells are human. In some embodiments, germ cells and somatic cells are human. In some embodiments, somatic cells are not human.

[0039] In some embodiments, germ cells and / or somatic cells are derived from stem cells. In some embodiments, germ cells and / or somatic cells are derived from stem cells such as pluripotent stem cells (PSCs), embryonic stem cells, or induced pluripotent stem cells (iPSCs). In some embodiments, germ cells and / or somatic cells are derived from stem cells that are pluripotent stem cells. The methods herein can utilize a variety of sources of pluripotent stem cells, including embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs). In some embodiments, pluripotent stem cells are induced pluripotent stem cells (iPSCs) artificially derived from non-pluripotent cells. In some embodiments, non-pluripotent cells are cells with a lower capacity for self-renewal and differentiation than pluripotent stem cells. iPSCs can be created through a known process known as reprogramming, in which non-pluripotent cells are effectively "dedifferentiated" into an embryonic stem cell-like state by manipulating them to express genes such as OCT4, SOX2, and KLF4 (Takahashi and Yamanaka Cell (2006) 126: 663-76).

[0040] In some embodiments, germ cells and / or somatic cells are derived from embryonic stem cells. In some embodiments, germ cells and / or somatic cells are derived from iPSCs.

[0041] In some embodiments, germ cells and / or somatic cells are isolated from in vivo tissue. In some embodiments, germ cells and / or somatic cells are isolated from primary tissue. In some embodiments, germ cells and / or somatic cells are isolated from primary ovarian tissue. In some embodiments, germ cells (e.g., PGCs or PGCLCs) and / or somatic cells are isolated from primary ovarian tissue. In some embodiments, primary ovarian tissue is detached and maintained in culture.

[0042] A. Germ cells In some embodiments, germ cells may be any cells capable of differentiating into reproductive cells, including but not limited to primordial germ cells, primordial germ cell-like cells, oogonia, and / or oocytes, or cells at any developmental stage between any of the aforementioned. In some embodiments, germ cells are cultured together with any somatic cells described herein, such as those described in Section IB, to form a cell mixture, such as any of those described in Section IC. In some embodiments, the cell mixture aggregates to form ovarian organoids, and in some aspects, primary follicles.

[0043] In some embodiments, germ cells are human germ cells. In some embodiments, germ cells include primordial germ cells (PGCs) or primordial germ cell-like cells (PGCLCs).

[0044] In some embodiments, germ cells (e.g., PGCs or PGCLCs) are derived from stem cells, such as pluripotent stem cells (PSCs), embryonic stem cells, or induced pluripotent stem cells (iPSCs), as described in Section I. In some embodiments, germ cells (e.g., PGCs or PGCLCs) are derived from embryonic stem cells. In some embodiments, germ cells (e.g., PGCs or PGCLCs) are derived from iPSCs. In some embodiments, germ cells, such as PGCs or PGCLCs, are induced in vitro. Exemplary in vitro methods for producing PGCLCs or PGCs from iPSCs or ESCs include, for example, Vijayakumar et al., Nat Commun (2023) 14, 5690,; Irie et al., Cell (2015), 160(1-2):253-68; Irie et al., Methods Mol Biol (2017) 1463:217-226; Jo et al., eLife (2022) 11:e72811; Overeem et al., Cell Rep Methods (2023) 3(6):100488; Sasaki et al., Cell Stem Cell, 17(2):178-94; Esfahani et al., Nat Comm. (2024) 2;15(1):167; Chen et al., Nat Methods (2011) 8, Provided in 424-429, each of these disclosures is incorporated herein by reference in its entirety. In some embodiments, pluripotent stem cells are cultured in a medium containing various supplements (e.g., FGF, BMP2, BMP4, or WNT inhibitors) that drive the differentiation of the stem cells into germ cells (e.g., PGCs or PGCLCs). Following differentiation under culture, in vitro induced PGCs or PGCLCs can be isolated by mechanical detachment, dissociation, and / or isolation. In some embodiments, in vitro induced PGCs or PGCLCs can be isolated using methods such as fluorescence-activated cell sorting (FACS) or magnetically activated cell sorting (MACS).In some embodiments, in vitro induced germ cells (e.g., PGCs or PGCLCs) are isolated by FACS using CD38 as a cell marker.

[0045] In some embodiments, germ cells (e.g., PGCs or PGCLCs) are isolated from in vivo tissue, for example, from primary tissue. In some embodiments, germ cells (e.g., PGCs or PGCLCs) are isolated from primary ovarian tissue. Exemplary methods for obtaining PGCLCs or PGCs from ovarian tissue are provided, for example, in Hayashi et al., Nat Protoc (2013) 8(8):1513-24, the disclosure of which is incorporated herein by reference in its entirety.

[0046] In some embodiments, germ cells can be collected from in vivo tissue. In some embodiments, germ cells can be collected from fetal ovarian tissue. In some examples, germ cells are collected from fetal ovarian tissue of a fetus between 6 and 22 weeks of gestation. For example, bovine fetal ovarian tissue can be collected between 6 and 22 weeks of gestation, pig fetal ovarian tissue can be collected between 6 and 22 weeks of gestation, non-human primate fetal ovarian tissue can be collected between 6 and 22 weeks of gestation, and mouse fetal ovarian tissue can be collected at 10 to 21 days of gestation. Fetal tissue can be isolated and prepared by methods known in the art. For example, fresh tissue is generally divided by chopping, tearing, grinding, and / or collagenase digestion. Desired cells can be isolated from contaminated cells and material by washing, filtering, centrifugation, and / or picking procedures, and optionally cultured and / or cryopreserved as needed before encapsulation. In some embodiments, primary ovarian tissue is detached and maintained under culture. In some cases, after tissue detachment, the ovary is chemically and mechanically dissociated to obtain a single-cell suspension. After the single-cell suspension is generated, PGCs or PGCLCs can be isolated by mechanical detachment, dissociation, and isolation using protocols such as fluorescence-activated cell sorting (FACS) or magnetically activated cell sorting (MACS). In some embodiments, native germ cells (e.g., PGCs or PGCLCs) are isolated by FACS.

[0047] In some aspects, germ cells are isolated from in vivo tissue. In some aspects, germ cells are derived from stem cells. In some aspects, germ cells are a mixture of cells isolated from in vivo tissue and germ cells derived from stem cells. In some aspects, germ cells are not a mixture of cells isolated from in vivo tissue and germ cells derived from stem cells.

[0048] In some embodiments, different germ cell populations may be defined and / or identified by any suitable marker (e.g., an expressed gene or reporter). In some embodiments, germ cells may be defined and / or identified by primordial germ cell marker genes. In some embodiments, germ cells express one or more primordial germ cell marker genes. Examples of primordial germ cell marker genes include, but are not limited to, NANOS3, TFAP2C, SOX17, PRDM1, POU5F1, OCT4, NANOG, TFCP2L1, and TBXT. In some embodiments, germ cells express one or more of the NANOS3, TFAP2C, SOX17, PRDM1, POU5F1, NANOG, and TBXT primordial germ cell marker genes. In some embodiments, germ cells express one or more of the TFAP2C, PRDM1, and POU5F1 primordial germ cell marker genes. In some embodiments, germ cells express the primordial germ cell marker genes NANOS3, TFAP2C, SOX17, PRDM1, POU5F1, NANOG, and TBXT. In some embodiments, germ cells express one or more of NANOS3, TFAP2C, SOX17, PRDM1, POU5F1, NANOG, and TBXT, and at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, and 97% of germ cells. %, or 98%, or at least about 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% express one or more of NANOS3, TFAP2C, SOX17, PRDM1, POU5F1, NANOG, and TBXT. In some embodiments, germ cells express the TFAP2C, PRDM1, and POU5F1 primordial germ cell marker genes.In some embodiments, at least about 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of germ cells express TFAP2C, PRDM1, and POU5F1. In some embodiments, at least about 80% of germ cells express TFAP2C, PRDM1, and POU5F1. In some embodiments, at least about 90% of germ cells express TFAP2C, PRDM1, and POU5F1. In some embodiments, at least about 95% of germ cells express TFAP2C, PRDM1, and POU5F1.

[0049] In some embodiments, germ cells do not express alternative lineage markers. Exemplary alternative lineage markers include, but are not limited to, FOXA2, HHEX, CDX2, and SOX2. In some embodiments, germ cells do not express one or more of the alternative lineage markers FOXA2, HHEX, CDX2, and SOX2. In some embodiments, germ cells do not express FOXA2, HHEX, CDX2, and SOX2. In some embodiments, germ cells do not express one or more of FOXA2, HHEX, CDX2, and SOX2, and at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, and 97% of germ cells. %, or 98%, or at least about 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% do not express one or more of FOXA2, HHEX, CDX2, and SOX2. In some embodiments, germ cells do not express FOXA2 and SOX2. In some embodiments, at least about 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of germ cells do not express FOXA2 and SOX2. In some embodiments, at least about 50% of germ cells do not express FOXA2 and SOX2. In some embodiments, at least about 60% of germ cells do not express FOXA2 and SOX2. In some embodiments, at least about 70% of germ cells do not express FOXA2 and SOX2. In some embodiments, at least about 80% of germ cells do not express FOXA2 and SOX2. In some embodiments, at least about 90% of germ cells do not express FOXA2 and SOX2.In some embodiments, at least about 95% of germ cells do not express FOXA2 and SOX2.

[0050] In some embodiments, germ cells express one or more of NANOS3, TFAP2C, SOX17, PRDM1, POU5F1, NANOG, and TBXT, and / or do not express one or more of FOXA2 and SOX2. In some embodiments, germ cells express NANOS3, TFAP2C, SOX17, PRDM1, POU5F1, NANOG, and TBXT, and do not express FOXA2 and SOX2. In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of germ cells, or at least about 30%, 40%, 50%, 60%, 70%, 80% %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% express NANOS3, TFAP2C, SOX17, PRDM1, POU5F1, NANOG, and TBXT, and / or do not express FOXA2 and SOX2. In some embodiments, at least about 80% of germ cells express NANOS3, TFAP2C, SOX17, PRDM1, POU5F1, NANOG, and TBXT, and / or do not express FOXA2 and SOX2. In some embodiments, at least about 90% of germ cells express NANOS3, TFAP2C, SOX17, PRDM1, POU5F1, NANOG, and TBXT, and / or do not express FOXA2 and SOX2. In some embodiments, at least about 95% of germ cells express NANOS3, TFAP2C, SOX17, PRDM1, POU5F1, NANOG, and TBXT, and / or do not express FOXA2 and SOX2.

[0051] B. Somatic cells In some embodiments, somatic cells may be any cells that form the body of a multicellular organism other than gametes and / or germ cells, such as ovarian somatic cells. In some embodiments, ovarian somatic cells are mammalian. In some embodiments, ovarian somatic cells are fetal ovarian somatic cells. In some embodiments, fetal ovarian somatic cells may be of any suitable species that support the development of germ cells within the organoid. In some embodiments, somatic cells are cultured together with any germ cells provided herein, such as those described in Section IA, to form a cell mixture, such as any of those described in Section IC. In some embodiments, the cell mixture aggregates to form an ovarian organoid, a primary follicle in some aspects.

[0052] In some embodiments, the species of somatic cells is the same as that of germ cells (e.g., human germ cells and human somatic cells). The species of somatic cells does not have to be the same as that of germ cells (e.g., human germ cells and non-human somatic cells). For example, the species can be any suitable species, including humans, pigs, rabbits, cattle, mice, rats, donkeys, rabbits, non-human primates, or other species. Somatic cells can be derived from progenitor cell types (e.g., they can be derived in vitro from stem cells or other precursors). In other embodiments, somatic cells can be collected from in vivo tissues such as fetal ovarian tissue. For example, bovine fetal ovarian tissue can be collected at 6-22 weeks of gestation, pig fetal ovarian tissue can be collected at 6-22 weeks of gestation, non-human primate fetal ovarian tissue can be collected at 6-22 weeks of gestation, or mouse fetal ovarian tissue can be collected at 10-21 days of gestation.

[0053] In some embodiments, somatic cells are induced in vitro from stem cells. Exemplary in vitro methods for generating somatic cells from iPSCs or ESCs are provided, for example, in WO 2022 / 094628, WO 2022 / 2211054, and Yoshino et al., Science (2021) 373(6552) eabe0237, the disclosures thereof being incorporated herein by reference in their entirety. In some embodiments, pluripotent stem cells are cultured in a medium containing one or more supplements that drive the differentiation of stem cells into somatic cells. In some embodiments, pluripotent stem cells are cultured in at least one medium condition containing one or more supplements, including but not limited to activin A, CHIR, BMP4, follistatin, EGF, FGF, retinoic acid (RA), and / or FGF. In some aspects, the culture is a stepwise differentiation process that involves differentiating stem cells into various intermediates, such as neonatal mesoderm, intermediate mesoderm, and then ovarian somatic cells (e.g., gonadal somatic cells). In some aspects, the culture is a multi-step culture that passes through mesoderm and intermediate mesoderm progenitor cells. In some aspects, the culture may be a 2D culture. In some aspects, the culture may be a 3D culture.

[0054] In some embodiments, pluripotent stem cells are cultured in medium 1, medium 2, and / or medium 3, each containing one or more supplements, including but not limited to activin A, CHIR, BMP4, follistatin, EGF, FGF, RA, and / or FGF, for the production of somatic cells. In some embodiments, CHIR is used for culture to the mesoderm. In some embodiments, RA and one or more other molecules are used for culture to the intermediate mesoderm. In some embodiments, pluripotent stem cells, such as embryonic stem cells (ESCs), are cultured for a suitable time to produce somatic cells. Following differentiation under culture, in vitro derivative cells can be isolated by mechanical detachment, dissociation, and isolation using protocols such as fluorescence-activated cell sorting (FACS) or magnetically activated cell sorting (MACS).

[0055] Any cells in the ovarian organoid, such as ovarian germ cells or ovarian somatic cells, can be obtained or induced by any suitable means. For example, cells can be obtained by mechanical detachment, dissociation, and / or isolation using protocols such as fluorescence-activated cell sorting (FACS) or magnetically activated cell sorting (MACS). In some embodiments, cells can be induced from other cell types, for example, by differentiation from progenitor cells or by any suitable means. For example, cells can be produced in vitro from any other cells, for example, by activation of cell signaling pathways, overexpression of transcription factors, or by other suitable methods.

[0056] Somatic ovarian cell types may include one or more somatic ovarian cell types, such as intermediate mesoderm, coelomic epithelium, granulosa cells, and bipotential gonads. In some embodiments, ovarian somatic cells may include intermediate mesoderm or may include intermediate mesoderm features, such as the expression of the gene WT1. In some embodiments, ovarian somatic cells may include coelomic epithelium or may include coelomic epithelial features, such as the expression of one or more genes selected from WT1 and GATA4. In some embodiments, ovarian somatic cells may include bipotential gonads or may include bipotential gonad features, such as the expression of one or more genes selected from WT1, GATA4, LHX9, and NR5A1. In some embodiments, somatic cells may include granulosa cells or may include granulosa cell features, such as the expression of one or more genes selected from WT1, GATA4, LHX9, NR5A1, and FOXL2. In some embodiments, ovarian somatic cells express one or more genes selected from WT1 and GATA4. In some embodiments, ovarian somatic cells express WT1 and GATA4. In some embodiments, ovarian somatic cells express one or more genes selected from WT1, GATA4, LHX9, and NR5A1. In some embodiments, ovarian somatic cells express WT1, GATA4, LHX9, and NR5A1. In some embodiments, ovarian somatic cells express one or more genes selected from WT1, GATA4, LHX9, NR5A1, and FOXL2. In some embodiments, ovarian somatic cells express WT1, GATA4, LHX9, NR5A1, and FOXL2.

[0057] C. Cell mixtures and cultures of germ cells and somatic cells In some embodiments, germ cells and / or somatic cells are cultured together to form a cell mixture. Germ cells and / or somatic cells can be obtained by any suitable means, such as by any of the methods provided herein, for example, in Sections IA and IB. In some embodiments, the cell mixture comprising germ cells and somatic cells includes germ cells that differentiate into oogonia and / or oocytes. In some embodiments, germ cells and / or somatic cells are cultured in the cell mixture to generate follicles.

[0058] In some embodiments, germ cells and / or somatic cells are cultured together in a prescribed medium containing a low concentration of total protein or protein substitute (e.g., less than 3.5 mg / mL), such as any serum-free medium described herein, for example, in Section II or the Examples. In some of the embodiments provided, the prescribed medium contains protein or protein substitute, and the total concentration of protein or protein substitute in the prescribed medium is lower than the concentration of protein or protein substitute in a standard culture medium, such as one containing 10% fetal bovine serum (FBS). In some embodiments, the prescribed medium contains protein or protein substitute, and the total concentration of protein or protein substitute in the prescribed medium is less than 3.5 mg / mL. In some embodiments, culture in a prescribed medium containing a small amount of protein or protein substitute increases germ cell viability, proliferation, and differentiation (e.g., compared to culture in the same medium containing more than 3.5 mg / mL of total protein, or the same medium containing at least 10% fetal bovine serum).

[0059] In some embodiments of the provided method, germ cells and somatic cells can be mixed into a cell mixture and cultured for a suitable time in a prescribed medium containing less than 3.5 mg / mL of protein or protein substitute, thereby causing the cells to aggregate and form ovarian organoids.

[0060] In some cases, a cell mixture containing germ cells and somatic cells is mixed for culture in a germ cell-to-somatic cell ratio of 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20, or approximately 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20, or any ratio in between. In some embodiments, a cell mixture containing germ cells and somatic cells is mixed for culture in a germ cell-to-somatic cell ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, or approximately 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, or any ratio in between. In some embodiments, a cell mixture containing germ cells and somatic cells is mixed for culture in a germ cell-to-somatic cell ratio of 1:3 or approximately 1:3. In some embodiments, a cell mixture containing germ cells and somatic cells is mixed for culture in a germ cell-to-somatic cell ratio of 1:10 or approximately 1:10.

[0061] In some aspects, at least 5,000 or at least about 5,000 germ cells and somatic cells are combined, mixed, and cultured. In some aspects, the total number of germ cells and somatic cells mixed to form a cell mixture is about 5,000 to about 250,000 total cells. In some aspects, the total number of germ cells and somatic cells mixed to form a cell mixture is about 10,000 to about 225,000 total cells. In some aspects, the total number of germ cells and somatic cells mixed to form a cell mixture is about 15,000 to about 210,000 total cells. In some aspects, the total number of germ cells and somatic cells mixed to form a cell mixture is about 20,000 to about 200,000 total cells.

[0062] In some embodiments, a cell mixture (i.e., germ cells and / or somatic cells) is cultured for a period of time sufficient to allow aggregation. After aggregation, the aggregated cell mixture of germ cells and somatic cells may form ovarian organoids. In some embodiments, the period sufficient to allow aggregation is about 12 to about 64 hours, or any time in between. In some embodiments, the period is about 18 to about 52 hours, or any time in between. In some embodiments, the period is about 24 to about 48 hours, or any time in between. In some embodiments, the cell mixture is cultured for about 24 to about 48 hours, thereby causing the cells to aggregate and form ovarian organoids.

[0063] In some aspects, the percentage of germ cells in ovarian organoids is 0.1%-90%, 0.5%-90%, 1%-90%, 0.1%-80%, 0.5%-80%, 1%-80%, 0.1%-70%, 0.5%-70%, 1%-70%, 0.1%-60%, 0.5%-60%, 1%-60%, 0.1%-50%, 0.5%-50%, 1%-50%, 0.1%-40%, 0.5%-40%, 1%-40%, 0.1%-30%, 0.5%-30%, 1%-30%, 0.1%-20%, 0.5%-20%, 1%-20%, 0.1%-10% %, 0.5%~10%, or 1%~10%, or approximately 0.1%~90%, 0.5%~90%, 1%~90%, 0.1%~80%, 0.5%~80%, 1%~80%, 0.1%~70%, 0.5%~70%, 1%~70%, 0.1%~60%, 0.5%~60%, 1%~60%, 0.1 The percentages are %~50%, 0.5%~50%, 1%~50%, 0.1%~40%, 0.5%~40%, 1%~40%, 0.1%~30%, 0.5%~30%, 1%~30%, 0.1%~20%, 0.5%~20%, 1%~20%, 0.1%~10%, 0.5%~10%, or 1%~10%. In some embodiments, the percentage of germ cells in ovarian organoids is 1%~20% or 1%~50% of the total number of cells in the organoids. In some embodiments, the percentage of germ cells in ovarian organoids is 1%~20% of the total number of cells in the organoids. In some embodiments, the percentage of germ cells in ovarian organoids is 1%~50% of the total number of cells in the organoids.

[0064] The culture of germ cells and somatic cells into ovarian organoids may be carried out by any preferred means, such as any method provided herein. In some embodiments, ovarian organoids may be cultured in accordance with generally published methods for ovarian organoid culture, but the ovarian organoids may be cultured in a prescribed medium such as a serum-free medium containing low levels of protein or protein substitutes, such as less than 3.5 mg / mL total.

[0065] In some embodiments, ovarian organoids are transferred to a container suitable for maintenance or culture. In some embodiments, the container is suitable for three-dimensional (3D) culture. Without being bound by specific theories or mechanisms, 3D culture is more effective than two-dimensional (2D) culture in providing a scaffold for cell differentiation. Suitable 3D culture systems may include, for example, suspension 3D culture, e.g., suspension plates; 3D microwell culture, e.g., ultra-low adhesion multiwell plates; 3D culture on hydrophobic surfaces; rotational culture; static 3D suspension culture; or bioreactors. Suspension plates are commercially available, for example, the PERFECTA 3D suspension plate from Biospherix, Parish, NY. Ultra-low adhesion multiwell plates (sometimes also called non-adherent culture vessels) are also commercially available, for example, the AGGREWELL® ultra-low adhesion multiwell plate from Stemcell Technologies, Vancouver, Canada.

[0066] In some embodiments, the container is not treated to promote cell adhesion and growth. In some embodiments, the container is a standard tissue culture plate that is not treated to promote cell adhesion and growth. In some embodiments, the cells do not adhere or adhere substantially during culture. In some embodiments, the cells are cultured in suspension.

[0067] In some embodiments, the container is a multiwell plate. In some embodiments, the container is a multiwell plate which is a 96-well plate, a 24-well plate, or a 6-well plate.

[0068] The organoids can be maintained in an ultra-low adhesion plate in which the organoids can suspend, and the organoids are in a fully immersed suspension culture. In some embodiments, ovarian organoids are cultured in a fully immersed suspension culture. In some embodiments, ovarian organoids are cultured indefinitely in a fully immersed suspension culture. In some embodiments, the organoids can be transferred to a gas-liquid interface mesh filter 48 hours after aggregation. In other embodiments, the organoids can be transferred to a gas-liquid interface mesh filter after aggregation. In some embodiments, 1 to 10 or about 1 to 10 organoids per mesh filter are cultured in a tissue culture plate. In other embodiments, 5 to 7 or about 5 to 7 organoids per mesh filter are cultured in a tissue culture plate.

[0069] In several aspects, ovarian organoids can be evaluated for characteristics such as cellular composition and morphology by any preferred means, including those described herein and in the examples. For example, ovarian organoids can be analyzed at regular time intervals, including analyses performed by next-generation sequencing, histology, staining, and imaging.

[0070] In some embodiments, ovarian organoids are cultured in a prescribed medium, such as those described in Section II and the Examples. In some embodiments, the prescribed medium is serum-free and contains protein or protein substitutes, with a total protein or protein substitute concentration of less than 3.5 mg / mL. In some embodiments, ovarian organoids are cultured in a prescribed medium containing less than 3.5 mg / mL of total protein or protein substitutes until oocytes and / or oogonia are induced. In some embodiments, ovarian organoids are further cultured until follicles are formed. In some embodiments, the further culture step is performed in the prescribed medium or a different medium. In some embodiments, culture in a prescribed medium increases germ cell survival, proliferation, and differentiation, supporting the generation of oocytes, oogonia, and / or follicles (e.g., primordial follicles) in the ovarian organoid culture.

[0071] 1. Oogonia and / or oocytes In some embodiments, germ cells differentiate into other germ cell types under culture conditions. For example, germ cells may be oogonia and / or oocytes, or may differentiate into oogonia and / or oocytes. In some embodiments, culturing germ cells in the cell mixture described in Section IC as part of the organoid culture described above induces the differentiation of germ cells into oogonia and / or oocytes. In some embodiments, culturing germ cells and somatic cells, as described, produces oogonia and / or oocytes.

[0072] For example, germ cells may be or can differentiate into oogonia. In some aspects, oogonia are germ cells that can differentiate into primary oocytes. In some aspects, oogonia express one or more genes selected from DDX4, DAZL, STRA8, SYCP3, and SYCP1. In some embodiments, germ cells may be or can differentiate into oocytes. In some embodiments, oocytes express one or more genes selected from FIGLA and ZP3.

[0073] In some embodiments, germ cells in the cell mixture express one or more oogonia markers for meiotic transition, e.g., SYCP3 and SYCP1. In some embodiments, germ cells express SYCP3 or SYCP1, and in some embodiments, germ cells express SYCP3 and SYCP1. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the germ cells, or a small percentage. At least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% express SYCP3 and / or SYCP1. In some embodiments, at least about 1% of germ cells express SYCP3 and SYCP1. In some embodiments, at least about 5% of germ cells express SYCP3 and SYCP1. In some embodiments, at least about 10% of germ cells express SYCP3 and SYCP1. In some embodiments, at least about 20% of germ cells express SYCP3 and SYCP1. In some embodiments, at least about 30% of germ cells express SYCP3 and SYCP1. In some embodiments, at least about 40% of germ cells express SYCP3 and SYCP1. In some embodiments, at least about 80% of germ cells express SYCP3 and SYCP1. In some embodiments, at least about 90% of germ cells express SYCP3 and SYCP1. In some embodiments, at least about 95% of germ cells express SYCP3 and SYCP1.

[0074] In some embodiments, germ cells in a cell mixture express DDX4. In some embodiments, germ cells express DDX4. In some embodiments, at least about 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the germ cells express DDX4. In some embodiments, at least about 80% of the germ cells express DDX4. In some embodiments, at least about 90% of the germ cells express DDX4. In some embodiments, at least about 95% of the germ cells express DDX4.

[0075] In some embodiments, germ cells in a cell mixture express DAZL. In some embodiments, germ cells express DAZL. In some embodiments, at least about 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the germ cells express DAZL. In some embodiments, at least about 80% of the germ cells express DAZL. In some embodiments, at least about 90% of the germ cells express DAZL. In some embodiments, at least about 95% of the germ cells express DAZL.

[0076] As development progresses, oogonia begin to differentiate into oocytes, which then arrest at meiosis I. In some embodiments, oocytes express one or more genes selected from FIGLA, NOBOX, LMOD3, and ZP3.

[0077] In some embodiments, oocytes in a cell mixture express one or more of the genes FIGLA, NOBOX, LMOD3, and ZP3. In some embodiments, oocytes express one or more of the genes FIGLA, NOBOX, LMOD3, and ZP3. In some embodiments, at least about 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the oocytes express one or more of the genes FIGLA, NOBOX, LMOD3, and ZP3. In some embodiments, at least about 80% of the oocytes express one or more of the genes FIGLA, NOBOX, LMOD3, and ZP3. In some embodiments, at least about 90% of the oocytes express one or more of the genes FIGLA, NOBOX, LMOD3, and ZP3. In some embodiments, at least about 95% of the oocytes express one or more of the genes FIGLA, NOBOX, LMOD3, and ZP3.

[0078] 2. Primordial follicle In some embodiments, a cell mixture is cultured as part of an ovarian organoid and cultured until follicles are formed. In some embodiments, the method includes a step of further culturing oogonia and / or oocytes to generate primordial follicles. In some embodiments, the further culturing step is carried out in the same or a different medium as the prescribed medium.

[0079] In vivo, during the ovulatory cycle, primordial follicles develop into primary follicles, secondary follicles, antral follicles, and Graafian follicles (mature follicles), triggering ovulation. In some embodiments, the methods provided enable the generation of follicles such as primordial follicles. In some embodiments, the primordial follicle comprises an oocyte surrounded and / or in contact with a plurality of granulosa cells. In some embodiments herein, the primordial follicle can be further activated to generate, for example, primary follicles, secondary follicles, antral follicles, and / or Graafian follicles. In some embodiments, the primordial follicle can be further activated to generate, for example, primary follicles.

[0080] In some aspects, ovarian organoids develop into primary follicles after a certain period of culture. In some aspects, the period of culture for primary follicle development is approximately 100 to approximately 160 days, or any time in between. In some aspects, the period of culture for primary follicle development is 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, or 160 days, or approximately 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, or 160 days, or any time in between. In some aspects, the period of culture for primary follicle development is approximately 110 to approximately 150 days, or any time in between. In some aspects, the period of culture for primary follicle development is approximately 120 to 140 days, or any time in between. In some aspects, the period of culture for primary follicle development is approximately 14 weeks, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks, or any time in between. In some aspects, the period of culture for primary follicle development is 17, 28, 39, or 20 weeks, or any time in between. In some aspects, the period of culture for primary follicle development is approximately 17 to 20 weeks. In some aspects, the period of culture for primary follicle development is approximately 19 weeks. In some aspects, the period of culture for primary follicle development is approximately 20 weeks.

[0081] In some embodiments, follicles are identified or characterized by their phenotype. In some embodiments, follicles are identified by any suitable marker. In some embodiments, follicles are identified by expressed genes or reporters. In some embodiments, follicles can be identified by any suitable marker that identifies germ cells (e.g., oocytes) and somatic cells. Markers for identifying follicles are known in the art and are described herein. For example, germ cells (e.g., oogonia and / or oocytes) may express DDX4, and somatic cells (e.g., granulosa cells) may express FOXL2, and DDX4 and / or FOXL2 can be used as markers for specific cell types.

[0082] In some embodiments, the follicle is DDX4+ and FOXL2+. In some embodiments, the follicle contains DDX4+ germ cells and FOXL2+ somatic cells. In some embodiments, the follicle is characterized or identified by the presence of DDX4+ germ cells surrounded by FOXL2+ somatic cells. In some embodiments, the follicle can be identified by morphological features. In some embodiments, the follicle exhibits a flattened morphology. In some embodiments, the follicle does not exhibit a flattened morphology. In some embodiments, a developed follicle can remain in culture indefinitely.

[0083] II. Standard Medium In some embodiments, standardized media are provided herein for use in connection with any of the methods provided herein, for example, to generate oogonia, oocytes, and / or primordial follicles in ovarian organoid culture. The standardized media are serum-free, such as fetal bovine serum (FBS). In some embodiments, the methods provided are improvements compared to published protocols for ovarian organoid culture. It has been found herein that low protein content in standardized media, such as serum-free media, is advantageous for promoting ovarian organoid culture.

[0084] In some embodiments, the standard medium is a serum-free medium containing protein or protein substitute and having a total concentration of protein or protein substitute less than 3.5 mg / mL. In some embodiments, the standard medium having a total concentration of protein or protein substitute less than 3.5 mg / mL supports improved survival, proliferation, and differentiation of germ cells in culture, for example, in ovarian organoids. In some embodiments, the standard medium having a total concentration of protein or protein substitute less than 3.5 mg / mL supports improved follicle formation, such as primordial follicles. In some embodiments, the improvement is compared to the same culture conditions except that the culture medium contains FBS (e.g., 10% (v / v) FBS) instead of protein or protein substitute. In some embodiments, the improvement is compared to culture conditions using media used in published protocols for ovarian organoid culture, such as a medium containing FBS (e.g., 10% (v / v) FBS). In some embodiments, follicular development in a prescribed medium containing less than 3.5 mg / mL of total protein or protein substitute is increased compared to follicular development in a prescribed medium containing more than 3.5 mg / mL, such as 4.8 or about 4.8 mg / mL. In some embodiments, primordial follicular development is increased by culturing in a prescribed medium containing 3.5 or about 3.5 mg / mL of total protein or protein substitute compared to primordial follicular development in a prescribed medium containing more than 3.5 mg / mL, such as 4.8 or about 4.8 mg / mL.

[0085] In some embodiments, the prescribed medium contains one or more of the components described for the basic medium or supplement medium described herein. In some embodiments, the prescribed medium comprises the basic medium and the supplement medium. In some examples, the prescribed medium comprises the basic medium and the supplement medium, and the supplement medium contains a protein or protein substitute. In some examples, the prescribed medium comprises the basic medium and the supplement medium, and the supplement medium contains a protein or protein substitute, and the concentration of the total protein or protein substitute in the prescribed medium is less than 3.5 mg / mL. In some embodiments, the prescribed medium contains the protein or protein substitute in the basic medium. In some embodiments, the prescribed medium contains the protein or protein substitute in the basic medium, and the protein or protein substitute is a protein. In some embodiments, the prescribed medium contains the protein or protein substitute in the basic medium, and the protein or protein substitute is a protein substitute. In some embodiments, the protein or protein substitute in the prescribed medium contains serum protein components. In some embodiments, the protein or protein substitute in the prescribed medium contains mammalian serum protein components. In some embodiments, the serum protein components are defined.

[0086] In some embodiments, the prescribed medium comprises a basic medium and contains a protein or protein substitute. In some examples, the prescribed medium comprises a basic medium and contains a protein or protein substitute, such as any of the proteins or protein substitutes provided herein, including any of the proteins or protein substitutes that may be included in Section II.B. In some embodiments, the prescribed medium contains the proteins or protein substitutes listed in Section II.B. In some embodiments, the prescribed medium contains a protein or protein substitute. In some embodiments, the concentration of the total protein or protein substitute in the prescribed medium is less than 3.5 milligrams (mg / mL) per milliliter. In some embodiments, the protein or protein substitute is albumin or an albumin substitute. In some embodiments, the albumin is human-derived albumin. In some embodiments, the albumin is recombinant albumin. In some embodiments, the albumin is natural human serum albumin. In some embodiments, the albumin is recombinant human serum albumin. In some embodiments, the albumin is recombinant albumin from a non-human source. In some embodiments, the protein or protein substitute is or contains lipid-enriched albumin. In some embodiments, the protein or protein substitute is or comprises lipid-enriched bovine serum albumin. The albumin substitute can be any protein or protein substitute source. Examples of such protein or protein substitute samples include, but are not limited to, bovine pituitary extract, plant hydrolysates (e.g., rice hydrolysate), bovine fetal albumin (fetuin), egg albumin, human serum albumin (HSA), or albumin derived from another animal, chick extract, bovine embryo extract, AlbumX® I lipid-rich bovine serum albumin, and AlbumX® II lipid-rich bovine serum albumin. In some embodiments, the protein or protein substitute comprises synthetic polymers.In some embodiments, the protein or protein substitute comprises polyvinyl alcohol (PVA) and / or polyvinylpyrrolidone (PVP). In some embodiments, the protein substitute refers to any compound that can substitute for a protein in a culture medium for use in the methods herein to give results substantially similar to those of a protein. In some embodiments, the protein substitute functions like a protein and / or has the physical properties of a protein.

[0087] In some embodiments, the standard medium includes a supplement medium. In some embodiments, the standard medium contains a specified serum substitute (DSR), optionally a specified knockout serum substitute (KSR), and / or AlbumX II. In some embodiments, the standard medium contains 0-15% (v / v) of a specified knockout serum substitute (KSR), and / or 0-15% (v / v) of AlbumX II. In some embodiments, the standard medium includes a supplement medium which is a specified serum substitute, optionally a specified knockout serum substitute (KSR).

[0088] In some embodiments, the prescribed medium is a prescribed serum-free or low-serum medium containing a dipeptide form of L-glutamine (e.g., L-alanyl-L-glutamine), a reducing agent (e.g., 2-mercaptoethanol), an antibiotic (e.g., penicillin-streptomycin), a vitamin (e.g., ascorbic acid), and at least one protein or protein substitute. In some embodiments, the standard medium contains 15% KnockOut® serum substitute (Gibco, catalog number: 10828028), 0.1 mM 2-ME (Fisher Chemical, catalog number: O3446I-100), 1x Glutamax® dipeptide supplement (Gibco, catalog number: 35050061), 1x Pen / Strep (Gibco, catalog number: 15070063), 0.1 mM NEAA (Gibco, catalog number: 11140050), 1 mM sodium pyruvate (Gibco, catalog number: 11360070), 10 μM Rock inhibitor (Biogems, catalog number: 1293823-10MG), and 100 μg / ml Contains Primocin (Invivogen, catalog number: ant-pm-1) and GMEM (Gibco, catalog number: 11710035).

[0089] In some embodiments, the prescribed medium is supplemented with at least one protein or protein substitute. In some embodiments, the prescribed medium contains one or more inorganic salts, sugars, and amino acids, as well as one protein or protein substitute, and optionally also contains vitamins, organic acids, antioxidants, and buffers. One or more additional supplements may be added, including one or more supplements containing at least one protein, such as a serum substitution protein, or one or more other components that support cell growth and expansion, so that the total protein or protein substitute in the medium is less than 3.5 mg / mL. In some embodiments, the at least one protein is a human protein or a recombinant protein. In some embodiments, the at least one protein is a human protein or a recombinant protein, such as a serum substitution protein, e.g., albumin. In some embodiments, the concentration of total protein and / or protein substitute in a prescribed medium containing protein and / or protein substitute is less than 3.5 mg / mL, less than 3.0 mg / mL, less than 2.5 mg / mL, less than 2.0 mg / mL, less than 1.5 mg / mL, less than 1.0 mg / mL, less than 0.5 mg / mL, or less than 0.1 mg / mL. In some embodiments, the concentration of total protein and / or protein substitute in a prescribed medium is about 0.1 mg / mL to 0.5 mg / mL, about 0.5 mg / mL to 1.0 mg / mL, about 1.0 mg / mL to 1.5 mg / mL, about 1.5 mg / mL to 2.0 mg / mL, about 2.0 mg / mL to 2.5 mg / mL, about 2.5 mg / mL to 3.0 mg / mL, or about 3.0 mg / mL to 3.5 mg / mL. In some embodiments, the concentration of total protein or protein substitute in the prescribed medium is 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 7.5% (v / v), or approximately 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 7.5% (v / v), or any of the values ​​mentioned above.In some embodiments, the concentration of total protein or protein substitute in the specified medium is 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, or 3.5 mg / mL, or approximately 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, or 3.5 mg / mL, or any of the values ​​mentioned above. In some embodiments, the total concentration of protein or protein substitute is less than or equal to the concentration of fetal bovine serum (FBS)-derived protein in a medium containing 7.5% (v / v) FBS. In some embodiments, the standard medium does not contain serum. In some embodiments, the standard medium does not contain FBS.

[0090] In some embodiments, the protein or protein substitute is contained in the supplement medium present in the standard medium. The standard medium contains the supplement medium containing the protein or protein substitute. In some embodiments, the supplement medium constitutes 7.5% (v / v) or less of the standard medium. In some embodiments, the supplement medium constitutes about 1% to 3%, about 3% to 5%, or about 5% to 7.5% (v / v) of the standard medium. In some embodiments, the supplement medium constitutes 0.5% or about 0.5% (v / v), 1.0% or about 1.0% (v / v), 1.5% or about 1.5% (v / v), 2.0% or about 2.0% (v / v), 2.5% or about 2.5% (v / v), 3.0% or about 3.0% (v / v), 3.5% or about 3.5% (v / v), 4.0% or about The supplement medium constitutes 4.0%(v / v), 4.5% or approximately 4.5%(v / v), 5.0% or approximately 5.0%(v / v), 5.5% or approximately 5.5%(v / v), 6.0% or approximately 6.0%(v / v), 6.5% or approximately 6.5%(v / v), 7.0% or approximately 7.0%(v / v), or 7.5% or approximately 7.5%(v / v), or any of the values ​​mentioned above. In some embodiments, the supplement medium constitutes 2%(v / v) of the standard medium.

[0091] A. Basic culture medium In some embodiments, the standard medium may include a basic medium to which other components are added to obtain the standard medium. In the embodiments herein, the basic medium does not contain serum. The basic medium may be any suitable basic medium. The basic medium may be a medium otherwise used for a wide variety of cell culture applications. In some embodiments, the basic medium contains non-essential amino acids. In some embodiments, the basic medium contains one or more amino acids. In some embodiments, the basic medium contains one or more of aspartic acid, glutamic acid, asparagine, serine, glutamine, histidine, glycine, threonine, arginine, alanine, tyrosine, cysteine, valine, methionine, norvaline, tryptophan, phenylalanine, isoleucine, leucine, lysine, hydroxyproline, sarcosine, and proline.

[0092] In some embodiments, the basal medium is artificial or synthetic medium. In some embodiments, the basal medium is an equilibrium salt solution (e.g., PBS, DPBS, HBSS, EBSS). In some embodiments, the basal medium is selected from Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle Basal Medium (BME), F-10, F-12, RPMI 1640, Glasgow Minimum Essential Medium (GMEM), Alpha Minimum Essential Medium (AlphaMEM), Advanced Minimum Essential Medium (AdvancedMEM), Iskov Modified Dulbecco Medium, and M199. In some embodiments, the basal medium is a compound medium (e.g., RPMI-1640, IMDM). In some embodiments, the basal medium is OpTmizer® CTS® T-Cell Expansion Basal Medium (ThermoFisher).

[0093] In some embodiments, the basic culture medium contains a nutrient mixture of inorganic salts, sugars, and amino acids, and optionally also contains vitamins, organic acids, antioxidants, and / or buffers.

[0094] In some embodiments, the basic medium contains CO3 and HCO3. In some embodiments, the CO3 / HCO3 content of the basic medium is balanced with gaseous CO2 (e.g., 5-10%), thereby maintaining an optimal pH in the medium. In some embodiments, the basic medium contains zwitterions such as HEPES. In some embodiments, the basic medium contains phenol red. In some embodiments, the basic medium does not contain phenol red.

[0095] In some embodiments, the basic culture medium contains inorganic salts. In some embodiments, the inorganic salts promote osmotic balance. In some embodiments, the inorganic salts regulate membrane potential by providing sodium, potassium, and calcium ions.

[0096] In some embodiments, the basic medium contains one or more carbohydrates. In some embodiments, the carbohydrates contain glucose. In some embodiments, the carbohydrates contain galactose. In some embodiments, the carbohydrates contain maltose. In some embodiments, the carbohydrates contain fructose.

[0097] In some embodiments, the basic medium contains fatty acids. In some embodiments, the basic medium contains lipids. In some embodiments, the basic medium contains vitamins (e.g., vitamin A, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin E). In some embodiments, the basic medium contains trace elements. In some embodiments, the trace elements contain copper. In some embodiments, the trace elements contain zinc. In some embodiments, the trace elements contain selenium. In some embodiments, the trace elements contain tricarboxylic acid intermediates.

[0098] In some embodiments, the basic medium contains a mixture of inorganic salts, sugars, amino acids, and optionally, vitamins, organic acids, and / or buffers or other well-known cell culture nutrients. In addition to providing nutrients, the basic medium may also help maintain pH and osmotic pressure. In some embodiments, reagents in the basic medium support cell growth, proliferation, and / or expansion. A wide variety of commercially available basic media are well known to those skilled in the art, including Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Iskov's Modified Dulbecco's Medium, and Hamm's Medium. In some embodiments, the basic medium is Iskov's Modified Dulbecco's Medium, RPMI-1640, Advanced-MEM, or α-MEM.

[0099] In some embodiments, the basic medium is protein-free. In some embodiments, the basic medium is human protein-free (e.g., human serum protein). In some embodiments, the basic medium is serum-free. In some embodiments, the basic medium is fetal bovine serum-free.

[0100] In some embodiments, the basic medium comprises Alpha MEM (Gibco, catalog number: 12571063) or Advanced MEM (Gibco 12492013). In some embodiments, the basic medium is further supplemented with 1x Glutamax L-glutamine (Gibco, catalog number: 35050061), 1x Pen / Strep (Gibco, catalog number: 15070063), 50 μg / ml ascorbic acid (Sigma, catalog number: 49752-10G), and 0.05-0.6 mM 2-ME (Fisher Chemical, catalog number: O3446I-100).

[0101] B. Supplement culture medium In some embodiments, the protein or protein substitute is contained in a supplement medium present in the standard medium. In some embodiments, the supplement medium is a standard serum substitute medium. In some embodiments, the supplement medium constitutes 7.5% or less of the standard medium. In some embodiments, the supplement medium constitutes approximately 1% to 3%, approximately 3% to 5%, or approximately 5% to 7.5% of the standard medium. In some embodiments, the supplement medium constitutes 0.5% or about 0.5%, 1.0% or about 1.0%, 1.5% or about 1.5%, 2.0% or about 2.0%, 2.5% or about 2.5%, 3.0% or about 3.0%, 3.5% or about 3.5%, 4.0% or about 4.0%, 4.5% or about 4.5%, 5.0% or about 5.0%, 5.5% or about 5.5%, 6.0% or about 6.0%, 6.5% or about 6.5%, 7.0% or about 7.0%, or 7.5% or about 7.5%, or any of the aforementioned values ​​of the standard medium. In some embodiments, the supplement medium constitutes 2% of the standard medium. In some embodiments, these and other supplements described herein are intended to be used as medium supplements (e.g., medium supplements for basic media). In some embodiments, the supplement is intended to be used as a supplement for cell maintenance, enlargement, and / or activation. In some embodiments, the supplement is intended to be used as a supplement for cell enlargement.

[0102] In some embodiments, the supplement medium contains a protein or protein substitute, and the protein or protein substitute is albumin or an albumin substitute. In embodiments herein, the standard medium containing the supplement medium contains a protein or protein substitute, and the total protein or protein substitute is less than 3.5 mg / mL.

[0103] In some embodiments, the supplement medium is a specified serum substitute medium. In some embodiments, the supplement medium may be any suitable substitute medium, such as those described in WO 1998 / 030679A1, which are incorporated herein in their entirety.

[0104] In some embodiments, the supplement medium comprises at least one protein or protein substitute. In some embodiments, the at least one protein or protein substitute is at least one mammalian serum protein component. In some embodiments, the protein or protein substitute is derived from an animal source. In some embodiments, the protein or protein substitute is not fetal bovine serum. In some embodiments, the at least one protein comprises albumin, transferrin, insulin, fibronectin, aprotinin, or fetuin. In some embodiments, the protein or protein substitute comprises one or more of albumin, insulin, and transferrin, optionally one or more of human or recombinant albumin, insulin, or transferrin.

[0105] In some embodiments, the protein or protein substitute in the supplement medium is albumin or an albumin substitute. In some embodiments, the protein in the supplement medium is albumin, which is human-derived albumin. In some embodiments, the albumin is recombinant albumin. In some embodiments, the albumin is natural human serum albumin. In some embodiments, the albumin is recombinant human serum albumin. In some embodiments, the albumin is recombinant albumin from a non-human source. The albumin substitute can be any protein or protein substitute source. Examples of protein or protein substitute samples include, but are not limited to, bovine pituitary gland extract, plant hydrolysates (e.g., rice hydrolysate), fetal bovine albumin (fetuin), egg albumin, human serum albumin (HSA), or albumin from another animal, chick extract, bovine embryo extract, AlbumX® I lipid-rich bovine serum albumin, and AlbumX® II lipid-rich bovine serum albumin. In some embodiments, the protein or protein substitute comprises a synthetic polymer. In some embodiments, the protein or protein substitute comprises polyvinyl alcohol (PVA) and / or polyvinylpyrrolidone (PVP). In some embodiments, the protein substitute is a compound that can substitute for or replace a protein in a culture medium for use in the methods described herein to give results substantially similar to those of a protein. In some examples, the protein substitute can function as a substrate for cell adhesion. In some embodiments, the prescribed medium contains a prescribed serum substitute, optionally a prescribed knockout serum substitute (KSR), and / or AlbumX® II lipid-rich bovine serum albumin. In some embodiments, the prescribed medium contains 0-15 (v / v)% of a prescribed knockout serum substitute (KSR) and / or 0-15% (v / v) AlbumX® II lipid-rich bovine serum albumin.

[0106] In some aspects, the standard medium contains a protein that is transferrin or a transferrin substitute. In aspects herein, if the supplement medium contains transferrin, the supplement medium also contains another protein or protein substitute, and the total concentration of the protein or protein substitute is less than 3.5 mg / mL, including transferrin. In some aspects, the transferrin or transferrin substitute is human transferrin or derived from human transferrin. In some aspects, the transferrin or transferrin substitute is derived from human serum or plasma. In some aspects, the transferrin or transferrin substitute is recombinant transferrin. In some aspects, the transferrin is iron-saturated transferrin. In some aspects, the transferrin is iron-saturated human transferrin. In some embodiments, the concentration of transferrin is such that, after the supplement is combined with the basic medium (such as those described herein), the concentration of transferrin in the medium is: 10 or about 10 mg / L to 50 or about 50 mg / L, 10 or about 10 mg / L to 100 or about 100 mg / L, 10 or about 10 mg / L to 150 or about 150 mg / L, 10 or about 10 mg / L to 200 or about 200 mg / L, 10 or about 10 mg / L to 250 or about 250 mg / L, 10 or about 10 mg / L to 300 or about 300 mg / L, 10 or about 10 mg / L to 350 or about 350 mg / L, 10 or about 10 mg / L to 400 or about 400 mg / L, 10 or about 10 mg / L to 450 or about 450 mg / L, 10 or about 10 mg / L ~ 500 or approximately 500 mg / L, 10 or approximately 10 mg / L ~ 550 or approximately 550 mg / L, 10 or approximately 10 mg / L ~ 600 or approximately 600 mg / L, 10 or approximately 10 mg / L ~ 650 or approximately 650 mg / L, 10 or approximately 10 mg / L ~ 750 or approximately 750 mg / L, 50 or approximately 50 mg / L ~ 100 or approximately 100 mg / L, 50 or approximately 50 mg / L ~ 150 or approximately 150 mg / L, 50 or approximately 50mg / L~200 or approximately 200 mg / L, 50 or approximately 50 mg / L~250 or approximately 250 mg / L, 50 or approximately 50 mg / L~300 or approximately 300 mg / L, 50 or approximately 50 mg / L~350 or approximately 350 mg / L, 50 or approximately 50 mg / L~400 or approximately 400 mg / L, 50 or approximately 50 mg / L~450 or approximately 450 mg / L, 50 or approximately 50 mg / L~500 or approximately 500 mg / L, 50 or approximately 50 mg / L~550 or approximately 550 mg / L, 50 or approximately 50 mg / L~600 or approximately 600 mg / L, 50 or approximately 50 mg / L~650 or approximately 650 mg / L, 50 or approximately 50 mg / L~750 or approximately 750 mg / L, 100 or approximately 100 mg / L~150 or approximately 150 mg / L, 100 or approximately 100 mg / L~200 or approximately 200 mg / L, 100 or approximately 100 mg / L~250 or approximately 250 mg / L, 100 or approximately 100 mg / L~300 or approximately 300 mg / L, 100 or approximately 100 mg / L~350 or approximately 350 mg / L, 100 or approximately 100 mg / L~400 or approximately 400 mg / L, 100 or approximately 100 mg / L~450 or approximately 450 mg / L, 100 or approximately 100 mg / L~500 or approximately 500 mg / L, 100 or approximately 100 mg / L~550 or approximately 550 mg / L, 100 or approximately 100 mg / L~600 or approximately 600 mg / L, 100 or approximately 100 mg / L~650 or approximately 650 mg / L, 100 or approximately 100 mg / L~750 or approximately 750 mg / L, 150 or approximately 150 mg / L~200 or approximately 200 mg / L, 150 or approximately 150 mg / L~250 or approximately 250 mg / L, 150 or approximately 150 mg / L~300 or approximately 300 mg / L, 150 or approximately 150 mg / L~350 or approximately 350 mg / L, 150 or approximately 150 mg / L~400 or approximately 400 mg / L, 150 or approximately 150 mg / L~450 or approximately 450 mg / L, 150 or approximately 150 mg / L~500 or approximately 500 mg / L, 150 or approximately 150 mg / L~550 or approximately 550 mg / L, 150 or approximately 150 mg / L ~ 600 or approximately 600mg / L, 150 or approximately 150 mg / L ~ 650 or approximately 650 mg / L, 150 or approximately 150 mg / L ~ 750 or approximately 750 mg / L, 200 or approximately 200 mg / L ~ 250 or approximately 250 mg / L, 200 or approximately 200 mg / L ~ 300 or approximately 300 mg / L, 200 or approximately 200 mg / L ~ 350 or approximately 350 mg / L, 200 or approximately 200 mg / L ~ 400 or approximately 400 mg / L, 200 or approximately 200 mg / L ~ 450 or approximately 450 mg / L, 200 or approximately 200 mg / L ~ 500 or approximately 500 mg / L, 200 or approximately 200 mg / L ~ 550 or approximately 550 mg / L, 200 or approximately 200 mg / L ~ 600 or approximately 600 mg / L, 200 or approximately 200 mg / L to 650 or approximately 650 mg / L, 200 or approximately 200 mg / L to 750 or approximately 750 mg / L, 250 or approximately 250 mg / L to 300 or approximately 300 mg / L, 250 or approximately 250 mg / L to 350 or approximately 350 mg / L, 250 or approximately 250 mg / L to 400 or approximately 400 mg / L, 250 or approximately 250 mg / L to 450 or approximately 450 mg / L, 250 or approximately 250 mg / L to 500 or approximately 500 mg / L, 250 or approximately 250 mg / L to 550 or approximately 550 mg / L, 250 or approximately 250 mg / L to 600 or approximately 600 mg / L, 250 or approximately 250 mg / L to 650 or approximately 650 mg / L, 250 or approximately 250 mg / L to 750 or approximately 750 mg / L, 300 or approximately 300 mg / L to 350 or approximately 350 mg / L, 300 or approximately 300 mg / L to 400 or approximately 400 mg / L, 300 or approximately 300 mg / L to 450 or approximately 450 mg / L, 300 or approximately 300 mg / L to 500 or approximately 500 mg / L, 300 or approximately 300 mg / L to 550 or approximately 550 mg / L, 300 or approximately 300 mg / L to 600 or approximately 600 mg / L, 300 or approximately 300 mg / L to 650 or approximately 650 mg / L, 300 or approximately 300 mg / L to 750 or approximately 750 mg / L, 350 or approximately 350 mg / L to 400 or approximately 400 mg / L, 350 or approximately 350 mg / L ~ 450 or approximately 450mg / L, 350 or approximately 350 mg / L ~ 500 or approximately 500 mg / L, 350 or approximately 350 mg / L ~ 550 or approximately 550 mg / L, 350 or approximately 350 mg / L ~ 600 or approximately 600 mg / L, 350 or approximately 350 mg / L ~ 650 or approximately 650 mg / L, 350 or approximately 350 mg / L ~ 750 or approximately 750 mg / L, 400 or approximately 400 mg / L ~ 450 or approximately 450 mg / L, 400 or approximately 400 mg / L ~ 500 or approximately 500 mg / L, 400 or approximately 400 mg / L ~ 550 or approximately 550 mg / L, 400 or approximately 400 mg / L ~ 600 or approximately 600 mg / L, 400 or approximately 400 mg / L ~ 650 or approximately 650 mg / L, 400 or approximately 400 mg / L to 750 or approximately 750 mg / L, 450 or approximately 450 mg / L to 500 or approximately 500 mg / L, 450 or approximately 450 mg / L to 550 or approximately 550 mg / L, 450 or approximately 450 mg / L to 600 or approximately 600 mg / L, 450 or approximately 450 mg / L to 650 or approximately 650 mg / L, 450 or approximately 450 mg / L to 750 or approximately 750 mg / L, 500 or approximately 500 mg / L to 550 or approximately 550 mg / L, 500 or approximately 500 mg / L to 600 or approximately 600 mg / L, 500 or approximately 500 mg / L to 650 or approximately 650 mg / L, 500 or approximately 500 mg / L to 750 or approximately 750 mg / L, 550 or approximately 550 mg / L ~ 600 or approximately 600 mg / L, 500 or approximately 500 mg / L ~ 650 or approximately 650 mg / L, 500 or approximately 500 mg / L ~ 750 or approximately 750 mg / L, 550 or approximately 550 mg / L ~ 600 or approximately 600 mg / L, 550 or approximately 550 mg / L ~ 650 or approximately 650 mg / L, 550 or approximately 550 mg / L ~ 750 or approximately 750 mg / L, 600 or approximately 600 mg / L ~ 650 or approximately 650 mg / L, 600 or approximately 600 mg / L ~ 750 or approximately 750 mg / L, or 650 or approximately 650 mg / L ~ 750 or approximately 750mg / L. In some embodiments, the concentration of transferrin is such that the concentration of transferrin in the medium is 100 or about 100 mg / L after the supplement is combined with the basal medium (such as those described herein). In some embodiments, the concentration of transferrin is such that the concentration of transferrin in the medium is 50 or about 50 mg / L to 150 or about 150 mg / L after the supplement is combined with the basal medium (such as those described herein). In embodiments herein, if the supplement medium contains transferrin, the supplement medium also contains another protein or protein substitute, and the total concentration of the protein or protein substitute, including transferrin, is less than 3.5 mg / mL.

[0107] In some embodiments, the prescribed medium contains a protein or protein substitute containing insulin or an insulin substitute (substitute).

[0108] In the embodiments herein, if the supplement medium contains insulin, the supplement medium also contains another protein or protein substitute, and the total concentration of the protein or protein substitute, including insulin, is less than 3.5 mg / mL. Numerous insulins are known to those skilled in the art. See Gilman, AG et al, Eds., The Pharmacological Basis of Therapeutics, Pergamon Press, New York, 1990, pp. 1463-1495. Any known insulin can be included in the standard medium or supplement medium, and the total protein concentration in the standard medium, including insulin, is less than 3.5 mg / mL. In some embodiments, insulin, rather than an insulin substitute, is used in the supplement medium and standard medium. In some embodiments, insulin is zinc insulin. In some embodiments, insulin is human zinc insulin.

[0109] In some embodiments, the supplement medium contains insulin that is human insulin or derived from human insulin. In some embodiments, the supplement medium contains insulin that is recombinant insulin. In some embodiments, the insulin is recombinant human insulin. In some embodiments, the concentration of insulin (or insulin substitute) is such that, after the supplement is combined with a basal medium (such as those described herein), the concentration of insulin (or insulin substitute) in the medium or around it is: about 1 mg / L to 2.5 or about 2.5 mg / L, 1 or about 1 mg / L to 5 or about 5 mg / L, 1 or about 1 mg / L to 7.5 or about 7.5 mg / L, 1 or about 1 mg / L to 10 or about 10 mg / L, 1 or about 1 mg / L to 12.5 or about 12.5 mg / L, 1 or about 1 mg / L to 15 or about 15 mg / L, 1 or about 1 mg / L to 17.5 or about 17.5 mg / L, 1 or about 1 mg / L to 20 or about 20 mg / L, 1 or about 1 mg / L to 22.5 or about 22.5 mg / L, 1 or about 1 mg / L to 25 or about 25 mg / L, 1 or about 1 mg / L ~ 27.5 or approximately 27.5 mg / L, 1 or approximately 1 mg / L ~ 30 or approximately 30 mg / L, 2.5 or approximately 2.5 mg / L ~ 5 or approximately 5 mg / L, 2.5 or approximately 2.5 mg / L ~ 7.5 or approximately 7.5 mg / L, 2.5 or approximately 2.5 mg / L ~ 10 or approximately 10 mg / L, 2.5 or approximately 2.5 mg / L ~ 12.5 or approximately 12.5 mg / L, 2.5 or approximately 2.5 mg / L ~ 15 or approximately 15 mg / L, 2.5 or approximately 2.5 mg / L ~ 17.5 or approximately 17.5 mg / L, 2.5 or approximately 2.5 mg / L ~ 20 or approximately 20 mg / L, 2.5 or approximately 2.5 mg / L ~ 22.5 or approximately 22.5 mg / L, 2.5 or approximately 2.5 mg / L ~ 25 or approximately 25 mg / L, 2.5 or approximately 2.5 mg / L ~ 27.5 or approximately 27.5 mg / L, 2.5 or approximately 2.5 mg / L ~ 30 or approximately 30 mg / L, 5 or approximately 5 mg / L ~ 7.5 or approximately 7.5 mg / L, 5 or approximately 5 mg / L ~ 10 or approximately 10 mg / L, 5 or approximately 5 mg / L ~ 12.5 or approximately 12.5 mg / L, 5 or approximately 5 mg / L to 15 or approximately 15 mg / L, 5 or approximately 5 mg / L to 17.5 or approximately 17.5 mg / L, 5 or approximately 5 mg / L to 20 or approximately 20 mg / L, 5 or approximately 5 mg / L to 22.5 or approximately 22.5 mg / L, 5 or approximately 5 mg / L to 25 or approximately 25 mg / L, 5 or approximately 5 mg / L to 27.5 or approximately 27.5 mg / L, 5 or approximately 5 mg / L to 30 or approximately 30 mg / L, 7.5 or approximately 7.5 mg / L to 10 or approximately 10 mg / L, 7.5 or approximately 7.5 mg / L to 12.5 or approximately 12.5 mg / L, 7.5 or approximately 7.5 mg / L to 15 or approximately 15 mg / L, 7.5 or approximately 7.5 mg / L to 17.5 or approximately 17.5 mg / L, 7.5 or about 7.5 mg / L ~ 20 or about 20 mg / L, 7.5 or about 7.5 mg / L ~ 22.5 or about 22.5 mg / L, 7.5 or about 7.5 mg / L ~ 25 or about 25 mg / L, 7.5 or about 7.5 mg / L ~ 27.5 or about 27.5 mg / L, 7.5 or about 7.5 mg / L ~ 30 or about 30 mg / L, 10 or about 10 mg / L ~ 12.5 or about 12.5 mg / L, 10 or about 10 mg / L ~ 15 or about 15 mg / L, 10 or about 10 mg / L ~ 17.5 or about 17.5 mg / L, 10 or about 10 mg / L ~ 20 or about 20 mg / L, 10 or about 10 mg / L ~ 22.5 or about 22.5 mg / L, 10 or about 10 mg / L ~ 25 or approximately 25 mg / L, 10 or approximately 10 mg / L ~ 27.5 or approximately 27.5 mg / L, 10 or approximately 10 mg / L ~ 30 or approximately 30 mg / L, 12.5 or approximately 12.5 mg / L ~ 15 or approximately 15 mg / L, 12.5 or approximately 12.5 mg / L ~ 17.5 or approximately 17.5 mg / L, 12.5 or approximately 12.5 mg / L ~ 20 or approximately 20 mg / L, 12.5 or approximately 12.5 mg / L ~ 22.5 or approximately 22.5 mg / L, 12.5 or approximately 12.5 mg / L ~ 25 or approximately 25 mg / L, 12.5 or approximately 12.5 mg / L ~ 27.5 or approximately 27.5 mg / L, 12.5 or approximately 12.5 mg / L ~ 30 or approximately 30 mg / L, 15 or approximately 15 mg / L ~ 17.5 or approximately 17.5 mg / L, 15 or approximately 15 mg / L to 20 or approximately 20 mg / L, 15 or approximately 15 mg / L to 22.5 or approximately 22.5 mg / L, 15 or approximately 15 mg / L to 25 or approximately 25 mg / L, 15 or approximately 15 mg / L to 27.5 or approximately 27.5 mg / L, 15 or approximately 15 mg / L to 30 or approximately 30 mg / L, 17.5 or approximately 17.5 mg / L to 20 or approximately 20 mg / L, 17.5 or approximately 17.5 mg / L to 22.5 or approximately 22.5 mg / L, 17.5 or approximately 17.5 mg / L to 25 or approximately 25 mg / L, 17.5 or approximately 17.5 mg / L to 27.5 or approximately 27.5 mg / L, 17.5 or approximately 17.5 mg / L to 30 or approximately 30 mg / L, 20 or about 20 mg / L to 22.5 or about 22.5 mg / L, 20 or about 20 mg / L to 25 or about 25 mg / L, 20 or about 20 mg / L to 27.5 or about 27.5 mg / L, 20 or about 20 mg / L to 30 or about 30 mg / L, 22.5 or about 22.5 mg / L to 25 or about 25 mg / L, 22.5 or about 22.5 mg / L to 27.5 or about 27.5 mg / L, 22.5 or about 22.5 mg / L to 30 or about 30 mg / L, 25 or about 25 mg / L to 27.5 or about 27.5 mg / L, or 27.5 or about 27.5 mg / L to 30 or about 30 mg / L. In some embodiments, the concentration of insulin or insulin substitute in the culture medium is 10 or about 10 mg / L. In some embodiments, the concentration of insulin or insulin substitute in the culture medium is 7.5 or approximately 7.5 mg / L to 12.5 or approximately 12.5 mg / L.

[0110] In some embodiments, the supplement medium contains hormones (e.g., growth hormone, insulin, hydrocortisone, triiodothyronine, estrogen, androgen, progesterone, prolactin, follicle-stimulating hormone, gastrin-releasing peptide). In some embodiments, the supplement medium contains alpha-globulin or beta-globulin.

[0111] In some embodiments, the supplement medium comprises peptides or peptide fractions (e.g., protein hydrolysates derived from animals, microorganisms, or plants).

[0112] In some embodiments, the concentration of total protein or protein substitute in the prescribed medium is less than 3.5 mg / mL, less than 3.0 mg / mL, less than 2.5 mg / mL, less than 2.0 mg / mL, less than 1.5 mg / mL, less than 1.0 mg / mL, less than 0.5 mg / mL, or less than 0.1 mg / mL. In some embodiments, the concentration of total protein or protein substitute in the prescribed medium is about 0.1 mg / mL to 0.5 mg / mL, about 0.5 mg / mL to 1.0 mg / mL, about 1.0 mg / mL to 1.5 mg / mL, about 1.5 mg / mL to 2.0 mg / mL, about 2.0 mg / mL to 2.5 mg / mL, about 2.5 mg / mL to 3.0 mg / mL, or about 3.0 mg / mL to 3.5 mg / mL (including values ​​at both ends). In some embodiments, the concentration of total protein or protein substitute in the prescribed medium is 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 7.5%, or approximately 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 7.5% (each v / v), or any of the values ​​mentioned above. In some embodiments, the concentration of total protein or protein substitute in the specified medium is 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, or 3.5 mg / mL, or approximately 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, or 3.5 mg / mL, or any of the values ​​mentioned above.

[0113] In some embodiments, the supplement medium comprises one or more additional components. In some embodiments, the one or more additional components in the supplement include vitamins. In some embodiments, the vitamins include fat-soluble vitamins (e.g., vitamin A, vitamin D, vitamin E, vitamin K). In some embodiments, the vitamins include water-soluble vitamins (e.g., B1, B2, B6, B1). 12 Contains C, folic acid, etc.

[0114] In some embodiments, one or more additional components include transferrin substitutes. In some embodiments, the transferrin substitute is a compound that can substitute for transferrin in the supplement to give results substantially similar to those of transferrin. Examples of transferrin substitutes include, but are not limited to, any iron chelate compounds. Examples of iron chelate compounds that can be used include, but are not limited to, iron chelates of ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), deferoxamine mesylate, dimercaptopropanol, diethylenetriaminepentaacetic acid (DTPA), and trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), as well as ferric citrate chelates and ferrous sulfate chelates.

[0115] In some embodiments, one or more additional components in the supplement medium include an insulin substitute that is not a protein. In some embodiments, the insulin substitute is a zinc-containing compound that can be used in place of insulin to produce results substantially similar to insulin. Examples of insulin substitutes include, but are not limited to, zinc chloride, zinc nitrate, zinc bromide, and zinc sulfate.

[0116] In some embodiments, one or more additional components include lipids. In some embodiments, the lipids include cholesterol. In some embodiments, the lipids include steroids. In some embodiments, the lipids include fatty acids (e.g., palmitic acid, stearic acid, oleic acid, linoleic acid). In some embodiments, the lipids include ethanolamine. In some embodiments, the lipids include choline. In some embodiments, the lipids include inositol.

[0117] In some embodiments, one or more additional components include a transition metal. In some embodiments, the transition metal includes iron. In some embodiments, the transition metal includes zinc. In some embodiments, the transition metal includes copper. In some embodiments, the transition metal includes chromium. In some embodiments, the transition metal includes iodine. In some embodiments, the transition metal includes cobalt. In some embodiments, the transition metal includes selenium. In some embodiments, the transition metal includes magnesium. In some embodiments, the transition metal includes molybdenum. In some embodiments, the supplement medium may be any preferred substitute medium, such as KnockOut® serum substitute. In some embodiments, the supplement medium includes one or more components selected from albumin or an albumin substitute, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the supplement medium comprises albumin or an albumin substitute, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements. In some embodiments, the supplement medium comprises transferrin (iron-saturated), insulin, lipid-rich albumin (AlbuMAX), one or more amino acids, one or more vitamins, one or more antioxidants, and one or more trace elements.In some embodiments, the supplement medium consists of transferrin (iron saturated), insulin, lipid-rich albumin (AlbuMAX), glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, ascorbic acid 2-PO4, Ag+, Al3+, Ba2+, Cd2+, Co2+, Cr3+, Ge4+, Se4+, Br-, I-, F-, Mn2+, Si4+, V5+, Mo6+, Ni2+, Rb+, Sn2+, and Zr4+.

[0118] Table 1 lists exemplary components of KnockOut® serum substitutes.

[0119] [Table 1]

[0120] III. Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or vocabulary used herein are intended to have the same meaning as that commonly understood by those skilled in the art in which the claimed subject matter relates. In some cases, terms that have a commonly understood meaning are defined herein for clarity and / or immediate reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from that commonly understood in the art.

[0121] As used herein, the term “approximately” refers to the normal range of error for each value, which is readily apparent to those skilled in the art. References to “approximately” values ​​or parameters herein include (and are described) aspects directed to the value or parameter itself.

[0122] As used herein, the singular forms “a,” “an,” and “the” refer to multiple objects unless the context explicitly indicates otherwise. For example, “a” or “an” means “at least one” or “one or more.”

[0123] Through this disclosure, various aspects of the claimed subject matter are presented in scope form. It should be understood that the scope form is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the claimed subject matter. Therefore, the scope description should be considered to specifically disclose not only the individual numerical values ​​within that range, but also all possible sub-ranges. For example, if a range of values ​​is provided, it should be understood that the upper and lower limits of that range, and each intervening value between them and any other stated or intervening values ​​within that range, are encompassed within the claimed subject matter. These smaller upper and lower limits may independently be included in smaller ranges, subject to any limits specifically excluded within the stated range, and are also encompassed within the claimed subject matter. If the stated range includes one or both limits, the range excluding one or both of these included limits is also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0124] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0125] As used herein, “optional” or “optionally” means whether the event or situation described thereafter occurs or does not occur, and that the description includes whether such event or situation occurs or does not occur. For example, “optionally substituted” means that the group is either unsubstituted or substituted.

[0126] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in mammals, often in humans. A pharmaceutical composition typically contains an effective amount of an active agent (e.g., cells, as amplified according to the method provided) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively.

[0127] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation adjuvant, or carrier, which is common in the art and used in conjunction with a therapeutic agent, forming part of a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and is compatible with the other components of the formulation. A pharmaceutically acceptable carrier is suitable for the formulation in which it is used.

[0128] The term "in vivo" refers to events that occur within the body of mammals.

[0129] The term "ex vivo" refers to an event occurring on or within tissues or cells of a mammalian subject, but outside the body of the mammalian subject. Typically, the event takes place in an external environment. In certain contexts, ex vivo procedures include those in which organs, cells, or tissues are taken from a subject, usually a living organism, for treatment or procedure, and then returned to the subject.

[0130] The term "in vitro" refers to events that occur in a laboratory or other experimental setting.

[0131] As used herein, a kit is a packaged combination that optionally includes other elements, such as additional reagents and instructions for use of the combination or its components.

[0132] The term “package insert” is used to refer to the instructions that are customarily included in the market packaging of a therapeutic product, which include information regarding indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings relating to the use of such therapeutic product.

[0133] As used herein, “product” refers to a product that is manufactured and, in some cases, available for sale. In some embodiments, this term may refer to a composition contained within a package, such as in a container.

[0134] It is understood that the aspects and embodiments of the present invention described herein include aspects and embodiments that "include," "consist of," and "essentially consist of." [Examples]

[0135] IV. Examples The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0136] Example 1: Preparation and analysis of organoid cultures Ovarian organoids containing in vitro induced primordial germ cells (PGCs) and ovarian somatic cells were established for long-term culture and analysis.

[0137] Human in vitro induced PGCs were induced, expanded, dissociated, and sorted from human pluripotent stem cells (hPSCs) according to published protocols (e.g., as described in Irie et al., 2015 and Sasaki et al., Cell Stem Cell, 17(2):178-94, 2015). The hPSCs contained a constitutive fluorescent reporter (PSC-derived fluorescent reporter) for subsequent visualization of the in vitro induced PGCs. For FACS isolation, the cultures were dissociated, PGCs were labeled with anti-CD38 fluorescent antibody (Biolegend 303511), and sorted by FACS to obtain isolated PGCs. The isolated PGCs were resuspended in agglutination medium at a concentration of approximately 5,000 cells / 100 μL.

[0138] The agglutination medium consisted of 15% KnockOut® serum substitute (Gibco, catalog number: 10828028), 0.1 mM 2-ME (Fisher Chemical, catalog number: O3446I-100), 1x Glutamax® dipeptide supplement (Gibco, catalog number: 35050061), 1x Pen / Strep (Gibco, catalog number: 15070063), 0.1 mM NEAA (Gibco, catalog number: 11140050), 1 mM sodium pyruvate (Gibco, catalog number: 11360070), 10 μM Rock inhibitor (Biogems, catalog number: 1293823-10MG), and 100 μg / ml It consists of GMEM (Gibco, catalog number: 11710035) containing Primocin (Invivogen, catalog number: ant-pm-1).

[0139] Fetal ovarian somatic cells were collected by mechanical detachment and dissociated into single-cell suspensions according to standard procedures. The somatic cells were resuspended in agglutination medium at concentrations ranging from 45,000 to 180,000 cells / 100uL.

[0140] In vitro induced PGCs and somatic cells were mixed together in a ratio of 1:10 to 1:3 PGC:somatic cells and plated into the wells of a 96-well ultra-low adhesion U-bottom plate (Nexcelom, catalog number: ULA-96U) with 25,000 to 200,000 total cells per well. The cells were cultured by completely immersing them in aggregate suspension culture medium at 37°C and 5% CO2 for 48 hours to form cell aggregates (ovarian organoids or organoids). The cells were then transferred to long-term culture medium in the same plate, or onto Millicell Culture Plate Inserts (Thermo Fisher, catalog number: PICMO1250) immersed in long-term culture medium in a 24-well culture plate.

[0141] The long-term culture medium consisted of either Alpha MEM (Gibco, catalog number: 12571063) or Advanced MEM (Gibco 12492013), supplemented with 1x Glutamax (Gibco, catalog number: 35050061), 1x Pen / Strep (Gibco, catalog number: 15070063), 50 μg / ml ascorbic acid (Sigma, catalog number: 49752-10G), 0.05-0.6 mM 2-ME (Fisher Chemical, catalog number: O3446I-100), and various amounts of fetal bovine serum (FBS) (Corning, catalog number: MT35015CV), or was formulated as a standard medium containing a specified serum substitute (DSR) as described in the examples below. In the following examples, the standard medium is AlphaMEM supplement medium containing various concentrations of specified serum substitutes (DSRs) (e.g., KnockOut serum substitute, Gibco, catalog number 10828028). The medium was changed every 2-3 days.

[0142] Ovarian organoids were collected at regular time intervals for analysis, including next-generation sequencing, histology, staining, and imaging, as described below and in the examples below.

[0143] For live bright-field imaging, ovarian organoids were imaged at 4x and 10x magnification over the entire culture life using an EVOS M5000 (ThermoFisher).

[0144] For histological staining and analysis, organoids were harvested, washed once with PBS, and then fixed in 4% PFA (ThermoScientific J61984.AP) for 30 minutes. The organoids were then washed once with PBS and stored in 70% ethanol in a sterile aqueous solution. The organoids were processed with a Histocore Pegasus paraffin processor and embedded in paraffin wax blocks. The organoids were sectioned to a thickness of 3–5 μm, stained using routine immunofluorescence (e.g., as described in Miller et al., Stem Cell Reports, 2018), and imaged under a fluorescence microscope.

[0145] Example 2: Robust survival and proliferation of in vitro induced primordial germ cells in ovarian organoid cultures Ovarian organoids were prepared as described above in Example 1 and cultured in long-term medium containing 2% (v / v) FBS, 10% (v / v) FBS, or in long-term medium formulated as a standard medium containing 2% standardized serum substitute (DSR), 10% DSR, or in a mixture of the two long-term media, 2% (v / v) FBS / DSR (a 1:1 mixture of 2% (v / v) FBS long-term medium and 2% DSR long-term medium). After 21 days, organoids were fixed, sectioned, and stained to quantify the number of surviving PGCs per organoid. PGCs were derived from a fluorescently tagged PSC reporter strain and visualized by the expression of the PSC-derived fluorescent reporter and staining for AP2g to mark the PGCs. As shown in Figure 1, the condition using long-term medium, which is a standard medium containing 2% DSR, dramatically increased the survival of PGCs at day 21 compared to all other conditions.

[0146] Ovarian organoids were prepared as described above in Example 1 and cultured in long-term medium containing 2% DSR (normal medium) or 10% (v / v) FBS. Organoids were analyzed in live culture on days 7, 21, 43, and 79 by bright-field imaging (to visualize all cells) and fluorescence imaging (to visualize PSC-derived germ cells). As shown in Figure 2, PSCs under 2% DSR conditions (normal medium) showed brighter fluorescence on days 21, 43, and 79 compared to the 10% (v / v) FBS condition, and the brighter fluorescence indicates robust survival and proliferation of PSCs throughout the entire imaging time course. PSC survival and proliferation were dramatically improved on days 21, 43, and 79 compared to the 10% (v / v) FBS condition.

[0147] The results support the usefulness of using a standard medium with low protein concentrations for ovarian organoid culture, including supporting germ cell survival and proliferation.

[0148] Example 3: Production of oocytes from in vitro induced primordial germ cells in ovarian organoid cultures Ovarian organoids were prepared as described above in Example 1 and cultured in a standardized long-term medium containing 2% standardized serum substitute (DSR) or in a long-term medium containing 10% (v / v) FBS. After several weeks, the organoids were fixed, sectioned, and stained to assess the presence of germ cells competent to follicular aggregates, as evidenced by the expression of LMOD3 and ZP3 in germ cells expressing a fluorescent reporter. Human fetal ovarian sections were also fixed and stained as a positive control. As shown in Figure 3A, human fetal ovaries at 17 weeks of gestation contained germ cells competent to follicular aggregates co-expressing LMOD3 and ZP3. As shown in Figure 3B, ovarian organoids cultured in standardized long-term medium (standardized medium) containing 2% DSR showed a high density of germ cells competent to follicular aggregates, which expressed the PSC-derived fluorescent reporters, LMOD3 and ZP3. In contrast, as shown in Figure 3C, ovarian organoids cultured in long-term medium containing 10% (v / v) FBS showed little to no germ cells competent for follicular aggregation, and overall had far fewer PSC-derived reporter-expressing germ cells.

[0149] The results support the usefulness of using a standard medium with low protein concentrations in ovarian organoid culture, including supporting germ cell survival, proliferation, and differentiation into germ cells competent for follicular aggregation.

[0150] Example 4: Efficient production of primordial follicles in ovarian organoid cultures Ovarian organoids were prepared as described above in Example 1 and cultured in long-term medium (normal medium) containing 2% DSR or long-term medium containing 10% (v / v) FBS.

[0151] Several weeks later, organoids were fixed, sectioned, and stained to assess gene expression, cellular composition, and the presence of primordial follicles within the organoids. Organoids were stained and imaged for a PSC-derived reporter (marking germ cells), DDX4 (a marker for oogonia / oocytes expressed in the cytoplasm), and FOXL2 (a nuclear marker for ovarian somatic granulosa cells). Primordial follicles consist of a single DDX4+ oocyte surrounded by FOXL2+ somatic granulosa cells and may exhibit a flattened morphology.

[0152] Figures 4A and 4B show exemplary ovarian organoids from culture in long-term medium containing 2% DSR (standard medium) or long-term medium containing 10% (v / v) FBS. As shown in the figures, organoids cultured in 2% DSR showed robust expression of PSC-derived reporters, DDX4 and FOXL2, densely distributed throughout the organoids, indicating robust survival, proliferation, and differentiation of in vitro induced PGCs into oogonia / oocytes, with oogonia / oocytes mixed with somatic granulosa cells. In contrast, organoids cultured in 10% (v / v) FBS showed broad areas without cells, low survival of PSC-derived germ cells, and almost no FOXL2+ granulosa cells.

[0153] Figure 5 shows high-magnification images of ovarian organoids cultured in 2% DSR (standard medium), stained and imaged as in Figure 4 to assess the presence of primordial follicles. Human fetal ovarian sections were also fixed and stained as a positive control. Primordial follicles consist of a single DDX4+ oocyte surrounded by FOXL2+ somatic granulosa cells and may exhibit a flattened morphology. As shown in Figure 5, a human fetal ovary at 19 weeks of gestation showed dense primordial follicles characterized by DDX4+ cells surrounded by FOXL2+ granulosa cells. Similarly, ovarian organoids also showed dense primordial follicles, with PSC-derived reporter+ / DDX4+ germ cells surrounded by FOXL2+ cells. Individual follicles with characteristic follicular morphology can be clearly visualized within the organoids, as shown in several exemplary high-magnification panels.

[0154] The results support the usefulness of using a standard medium with low protein concentrations for ovarian organoid culture, including supporting germ cell survival, proliferation, differentiation, and follicular aggregation.

[0155] Example 5: Characterization of the intermediate population Human in vitro induced PGCLCs were induced, expanded, dissociated, and sorted from human pluripotent stem cells (hPSCs) according to a published protocol (e.g., as described in Irie et al., 2015 and Sasaki et al., Cell Stem Cell, 17(2):178-94, 2015), as described in Example 1. Ovarian organoids were prepared as described above in Example 1 and cultured in a standardized long-term medium (standard medium) containing 2% DSR.

[0156] Single-cell RNA sequencing was performed on control human fetal germ cells from 6 to 22 weeks post-conception (pwc), spanning developmental stages 1 to 4 (Figure 6A). The data were then merged with single-cell RNA sequencing data from human pluripotent stem cell-derived germ cells from primordial germ cell-like cells (PGCLCs) after initiation of culture in a cell mixture (Figure 6B) and after culture of ovarian organoids (Figure 6C). As seen in Figures 6A–6C, after culturing ovarian organoids in a standard medium containing 2% standardized serum substitute (e.g., 2% KSR), a subset of in vitro cultured human germ cells derived from pluripotent stem cells directly overlapped with human fetal germ cells spanning stages 1 to 4, indicating the progression of in vitro cultured germ cells in this culture system.

[0157] Figure 7 shows cultured ovarian organoids (lower panel) prepared according to the method described in Example 1 above, compared to human fetal ovarian controls (upper panel). As shown in the figure, germ cells (identified by positive DDX4 staining) in both human fetal ovarian controls and ovarian organoids show positive expression of nuclear SYP3+ staining, indicating that the germ cells have undergone meiosis. The results demonstrate that germ cells derived from human pluripotent stem cells cultured according to the described method can progress to meiosis in ovarian organoid culture.

[0158] The present invention is not intended to be limited to any particular disclosed embodiment, for example, provided to illustrate various aspects of the invention. Various modifications to the compositions and methods described herein will become apparent from the descriptions and teachings herein. Such modifications may be made without departing from the true scope and spirit of this disclosure and are intended to remain within the scope of this disclosure.

Claims

1. A method for generating primordial follicles, (a) A step of providing a cell mixture of germ cells and somatic cells under culture, and (b) A step of culturing the cell mixture in a specified medium for a certain period of time in order to generate primordial follicles. Includes, The method wherein the prescribed medium contains protein or a protein substitute, and the concentration of the total protein or protein substitute in the prescribed medium is less than 3.5 milligrams (mg / mL) per milliliter.

2. The method according to claim 1, wherein each primordial follicle comprises (i) an oocyte and (ii) a plurality of granulosa cells, wherein the plurality of granulosa cells are or are derived from somatic cells of (a).

3. A method for generating oogonia and / or oocytes, (a) A step of providing a cell mixture of germ cells and somatic cells under culture, and (b) A step of culturing the cell mixture in a specified medium for a certain period of time in order to produce oogonia and / or oocytes. Includes, The method wherein the prescribed medium contains protein or a protein substitute, and the concentration of the total protein or protein substitute in the prescribed medium is less than 3.5 milligrams (mg / mL) per milliliter.

4. The method according to claim 3, further comprising the step of culturing the oogonia and / or oocytes to generate primordial follicles.

5. The method according to claim 4, wherein the further culturing step is performed in the same or a different culture medium as the specified culture medium.

6. The method according to any one of claims 1 to 5, wherein the concentration of the total protein or protein substitute in the prescribed culture medium is less than 3.5 mg / mL, less than 3.0 mg / mL, less than 2.5 mg / mL, less than 2.0 mg / mL, less than 1.5 mg / mL, less than 1.0 mg / mL, less than 0.5 mg / mL, or less than 0.1 mg / mL.

7. The method according to any one of claims 1 to 6, wherein the concentration of the total protein or protein substitute in the prescribed culture medium is about 0.1 mg / mL to 0.5 mg / mL, about 0.5 mg / mL to 1.0 mg / mL, about 1.0 mg / mL to 1.5 mg / mL, about 1.5 mg / mL to 2.0 mg / mL, about 2.0 mg / mL to 2.5 mg / mL, about 2.5 mg / mL to 3.0 mg / mL, or about 3.0 mg / mL to 3.5 mg / mL.

8. The method according to any one of claims 1 to 7, wherein the concentration of the total protein or protein substitute in the specified culture medium is 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, or 3.5 mg / mL, or approximately 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, or 3.5 mg / mL, or any of the above.

9. The method according to any one of claims 1 to 8, wherein the prescribed medium is a serum-free medium that does not contain serum.

10. The method according to any one of claims 1 to 9, wherein the standard culture medium does not contain FBS.

11. The aforementioned culture medium One or more of inorganic salts, sugars, amino acids, vitamins, organic acids, antioxidants, and buffers; and Protein or protein substitute A method according to any one of claims 1 to 10, including the method described in any one of claims 1 to 10.

12. The method according to any one of claims 1 to 11, wherein the prescribed medium comprises a protein or protein substitute in the basic medium.

13. The method according to claim 12, wherein the basic medium comprises one or more of Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle Basal Medium (BME), F-10, F-12, RPMI 1640, Glasgow Minimum Essential Medium (GMEM), Alpha Minimum Essential Medium (AlphaMEM), Advanced Minimum Essential Medium (AdvancedMEM), Iskov Modified Dulbecco Medium, and M199.

14. The method according to any one of claims 1 to 13, wherein the protein or protein substitute is a protein.

15. The method according to any one of claims 1 to 14, wherein the protein or protein substitute is a protein substitute.

16. The method according to any one of claims 1 to 15, wherein the protein or protein substitute is albumin or an albumin substitute, or comprises the same.

17. The method according to any one of claims 1 to 16, wherein the protein or protein substitute comprises one or more components selected from bovine pituitary gland extract, plant hydrolysates (e.g., rice hydrolysates), albumin, chick extract, bovine embryo extract, bovine fetal albumin (fetuin), egg albumin, human serum albumin (HSA), albumin derived from other animals, and bovine serum albumin.

18. The method according to any one of claims 1 to 17, wherein the protein or protein substitute is or comprises lipid-enriched albumin.

19. The method according to any one of claims 1 to 18, wherein the protein or protein substitute is or comprises lipid-enriched bovine serum albumin.

20. The method according to any one of claims 1 to 19, wherein the protein or protein substitute is AlbumX® lipid-rich bovine serum albumin, optionally AlbumX® I lipid-rich bovine serum albumin, or AlbumX® II lipid-rich bovine serum albumin, or comprises the same.

21. The method according to any one of claims 1 to 20, wherein the prescribed culture medium contains a synthetic component.

22. The method according to any one of claims 1 to 21, wherein the protein or protein substitute comprises a synthetic polymer.

23. The method according to claim 22, wherein the synthetic polymer is polyvinyl alcohol (PVA) and / or polyvinylpyrrolidone (PVP).

24. The method according to any one of claims 12 to 23, wherein the standard medium is prepared by adding the protein or protein substitute to the basic medium.

25. The method according to any one of claims 12 to 24, wherein the protein or protein substitute is provided by a supplement medium added to the basic medium.

26. The method according to claim 25, wherein the supplement medium is a specified serum substitute medium.

27. The method of 25 or 26, wherein the supplement medium is added to the basic medium until the final concentration is 7.5% or less.

28. The method according to any one of claims 25 to 27, wherein the supplement medium is added to the basic medium to a final concentration (v / v) of approximately 1% to 7.5%, approximately 1% to 5%, approximately 1% to 3%, approximately 3% to 5%, or approximately 5% to 7.5%.

29. The method according to any one of claims 25 to 28, wherein the supplement medium is added to the basic medium until the final concentration is 0.5% or about 0.5%, 1.0% or about 1.0%, 1.5% or about 1.5%, 2.0% or about 2.0%, 2.5% or about 2.5%, 3.0% or about 3.0%, 3.5% or about 3.5%, 4.0% or about 4.0%, 4.5% or about 4.5%, 5.0% or about 5.0%, 5.5% or about 5.5%, 6.0% or about 6.0%, 6.5% or about 6.5%, 7.0% or about 7.0%, or 7.5% or about 7.5%, or any of the above values.

30. The method according to any one of claims 25 to 27, wherein the supplement medium is added to the basic medium until it reaches a final concentration of 2%.

31. The method according to any one of claims 25 to 30, wherein the supplement medium comprises one or more components selected from the group consisting of albumin or an albumin substitute, one or more amino acids, one or more vitamins, one or more transferrins or transferrin substitutes, one or more antioxidants, one or more insulins or insulin substitutes, one or more collagen precursors, and one or more trace elements.

32. The method according to any one of claims 25 to 31, wherein the supplement culture medium comprises albumin or an albumin substitute.

33. The method according to any one of claims 11 to 32, wherein the antioxidant is selected from the group consisting of reduced glutathione, ascorbic acid, and ascorbic acid-2-phosphate.

34. The method according to any one of claims 31 to 33, wherein the collagen precursor is selected from the group consisting of L-proline and its polymers or derivatives, L-hydroxyproline and its polymers or derivatives, and ascorbic acid or its polymers.

35. The method according to any one of claims 31 to 34, wherein the transferrin substitute is an iron chelate selected from the group consisting of ferric citrate chelate and ferrous sulfate chelate, and optionally ferrous sulfate-7-hydrate-EDTA.

36. The method according to any one of claims 31 to 35, wherein the insulin substitute is selected from the group consisting of zinc chloride, zinc bromide, and zinc sulfate-7 water.

37. The method according to any one of claims 31 to 36, wherein the amino acid component comprises one or more amino acids selected from the group consisting of glycine, L-alanine, L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-glutamine, L-arginine, L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, and derivatives thereof.

38. where the trace element component is Ag + Al3 + Ba2 + Cd 2+ Co 2+ Cr 3+ Ge 4+ Se 4+ Br - I - Mn 2+ F - Si 4+ V 5+ Mo 6+ Ni 2+ Rb + Sn 2+ and one or more trace element moieties selected from the group consisting of Zr 4+ The method according to any one of claims 31 to 37, comprising

39. The aforementioned supplement medium contains lipid-rich albumin (AlbuMAX), L-glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, thiamine, reduced glutathione, L-ascorbic acid-2-phosphate, iron-saturated transferrin, insulin, sodium selenite, and Ag. + , Al3 + , Ba2 + , Cd 2+ Co 2+ , Cr 3+ , Ge 4+ , Se 4+ , Br - , I - Mn 2+ F - Si 4+ , V 5+ Mo 6+ Ni 2+ , Rb + Sn 2+ , and Zr 4+ A method according to any one of claims 25 to 38, including the method described above.

40. The method according to any one of claims 25 to 39, wherein the supplement medium is a knockout serum substitute (KSR).

41. The method according to any one of claims 1 to 40, wherein the prescribed medium is a basic medium supplemented with 1 to 5% of a prescribed knockout serum substitute (KSR).

42. The method according to any one of claims 1 to 41, wherein the cell mixture is an ovarian organoid.

43. The method according to claim 42, wherein the ovarian organoid is cultured in a fully immersed suspension culture.

44. The method according to claim 42 or 43, wherein the ovarian organoid is cultured at a gas-liquid interface using a permeable culture membrane.

45. The method according to any one of claims 1 to 44, further comprising the step of removing the prescribed medium from the cell mixture after oogonia and / or oocytes have been produced.

46. The method according to any one of claims 42 to 45, wherein the ovarian organoid is composed of 500 to 300,000 cells.

47. The method according to any one of claims 42 to 46, wherein the percentage of germ cells in the ovarian organoid is 1% to 20% or 1% to 50% of the total number of cells in the organoid.

48. The method according to any one of claims 1 to 47, wherein the cell mixture is cultured in vitro for 1 to 300 days.

49. The method according to any one of claims 1 to 48, wherein the period for generating primordial follicles is approximately 100 to 160 days, approximately 110 to 150 days, or approximately 120 to 140 days in culture.

50. The method according to any one of claims 1 to 49, wherein the period for generating primordial follicles is approximately 110 to approximately 150 days under culture.

51. The method according to any one of claims 1 to 50, wherein the period for generating primordial follicles is approximately 120 to 140 days under culture.

52. The method according to any one of claims 1, 2, and 4 to 51, further comprising the step of activating the primordial follicle to generate a primary follicle.

53. The method according to any one of claims 1, 2, and 4-52, further comprising the step of activating the primordial follicle to generate primary follicles, secondary follicles, antral follicles, and / or Graafian follicles.

54. The method according to any one of claims 1 to 53, wherein the germ cells are primordial germ cells (PGCs).

55. The method according to any one of claims 1 to 53, wherein the germ cells are primordial germ cell-like cells (PGCLCs).

56. At least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the germ cells, or at least about 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84% The method according to claim 54 or 55, wherein %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% express one or more primordial germ cell marker genes optionally selected from NANOS3, SOX17, NANOG, TBXT, TFAP2C, PRDM1, and POU5F1.

57. The method according to claim 54 or 55, wherein the germ cells express one or more primordial germ cell marker genes optionally selected from NANOS3, SOX17, NANOG, TBXT, TFAP2C, PRDM1, and POU5F1.

58. The method according to any one of claims 54 to 57, wherein the germ cells express one or more primordial germ cell marker genes optionally selected from TFAP2C, PRDM1, and POU5F1.

59. The method according to any one of claims 54 to 58, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the germ cells do not express one or more surrogate lineage markers.

60. The method according to any one of claims 54 to 59, wherein the germ cells do not express one or more surrogate lineage markers.

61. The method according to any one of claims 54 to 60, wherein the germ cells do not express one or more alternative strain markers optionally selected from FOXA2, HHEX, CDX2, and SOX2.

62. The method according to any one of claims 1 to 61, wherein the germ cells are, optionally, mammals, humans, non-human primates, pigs, cattle, or horses.

63. The method according to any one of claims 1 to 62, wherein the germ cells are derived from in vivo tissue.

64. The method according to any one of claims 1 to 63, wherein the germ cells are derived from stem cells, optionally pluripotent stem cells (PSCs), optionally induced pluripotent stem cells (iPSCs), or embryonic stem cells (ESCs).

65. The method according to any one of claims 1 to 64, wherein the germ cells differentiate into oogonia and / or oocytes.

66. The method according to any one of claims 3 to 65, wherein the oogonia express one or more oogonia marker genes, optionally selected from DDX4, DAZL, STRA8, and ZGLP1, and / or the germ cells express one or more oogonia markers for meiotic transition, optionally selected from SYCP3 and SYCP1.

67. Of the oogonia, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%, or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, or 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% express one or more oogonia marker genes, optionally selected from DDX4, DAZL, STRA8, and ZGLP1, and / or the reproductive At least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the cells, or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 1 The method according to any one of claims 3 to 65, wherein 0%, 11%, 12%, 13%, 14%, 15%, 16%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% express one or more oogonia markers for meiotic transition, optionally selected from SYCP3 and SYCP1.

68. The method according to any one of claims 3 to 65, wherein at least 1% or at least about 1% of the oogonia express one or more oogonia marker genes optionally selected from DDX4, DAZL, STRA8, and ZGLP1, and / or at least 1% or at least about 1% of the germ cells express one or more oogonia markers for meiotic transition optionally selected from SYCP3 and SYCP1.

69. The method according to any one of claims 3 to 65, wherein at least 5% or at least about 5% of the oogonia express one or more oogonia marker genes optionally selected from DDX4, DAZL, STRA8, and ZGLP1, and / or at least 5% or at least about 5% of the germ cells express one or more oogonia markers for meiotic transition optionally selected from SYCP3 and SYCP1.

70. The method according to any one of claims 2 to 69, wherein the oocyte expresses one or more oocyte marker genes optionally selected from FIGLA, ZP3, and LMOD3.

71. The method according to any one of claims 2 to 70, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the oocytes express one or more oocyte marker genes optionally selected from FIGLA, ZP3, and LMOD3.

72. The method according to any one of claims 2 to 71, wherein at least 60% or at least about 60% of the oocytes express one or more oocyte marker genes optionally selected from FIGLA, ZP3, and LMOD3.

73. The method according to any one of claims 2 to 72, wherein at least 70% or at least about 70% of the oocytes express one or more oocyte marker genes optionally selected from FIGLA, ZP3, and LMOD3.

74. The method according to any one of claims 2 to 73, wherein at least 80% or at least about 80% of the oocytes express one or more oocyte marker genes optionally selected from FIGLA, ZP3, and LMOD3.

75. The method according to any one of claims 2 to 74, wherein at least 90% or at least about 90% of the oocytes express one or more oocyte marker genes optionally selected from FIGLA, ZP3, and LMOD3.

76. The method according to any one of claims 1 to 75, wherein the somatic cells are mammalian ovarian somatic cells.

77. The method according to any one of claims 1 to 76, wherein the somatic cells are primary cells of mammalian origin.

78. The method according to any one of claims 1 to 77, wherein the somatic cells are primary cells derived from the ovary of a mammalian fetus.

79. The method according to any one of claims 1 to 78, wherein the somatic cells are derived from humans, non-human primates, pigs, rabbits, cattle, mice, rats, donkeys, and / or rabbits.

80. The method according to any one of claims 1 to 79, wherein the somatic cells are differentiated in vitro from another cell type.

81. The method according to any one of claims 1 to 80, wherein the somatic cells are differentiated from stem cells, optionally pluripotent stem cells (PSCs), optionally induced pluripotent stem cells (iPSCs), or embryonic stem cells (ESCs).

82. The method according to any one of claims 1 to 81, wherein the somatic cells include one or more ovarian somatic cell types.

83. The method according to any one of claims 1 to 82, wherein one or more of the somatic cell types express one or more genes characteristic of the intermediate mesoderm, including WT1.

84. The method according to any one of claims 1 to 83, wherein one or more of the somatic cell types express one or more genes characteristic of coelomic epithelium, including WT1 and GATA4.

85. The method according to any one of claims 1 to 84, wherein one or more of the somatic cell types express one or more genes characteristic of granulosa cells, including WT1, GATA4, LHX9, NR5A1, and / or FOXL2.

86. The method according to any one of claims 1 to 85, wherein one or more of the somatic cell types express one or more genes characteristic of bipotent gonads, including WT1, GATA4, LHX9, and / or NR5A1.

87. A composition comprising a primordial follicle produced by the method described in any one of claims 1 to 86.

88. Ovarian organoids produced by the method described in any one of claims 46 to 74.

89. An ovarian organoid comprising one or more primordial follicles, wherein each primordial follicle comprises (i) an oocyte differentiated from mammalian pluripotent stem cells under culture, and (ii) a plurality of granulosa cells.

90. The ovarian organoid according to claim 89, wherein the pluripotent stem cells are derived from humans, non-human primates, pigs, cattle, or horses.

91. The ovarian organoid according to claim 89 or 90, comprising at least 5 primordial follicles, at least 10 primordial follicles, at least 20 primordial follicles, at least 50 primordial follicles, at least 100 primordial follicles, at least 500 primordial follicles, or more than 500 primordial follicles.

92. An ovarian organoid derived from a cell mixture of ovarian germ cells and somatic cells under culture, and comprising at least 5, at least 10, at least 50, at least 100, or at least 500 oocytes and / or oogonia derived from said ovarian germ cells, wherein the ovarian germ cells are of human or non-human primate origin.

93. The ovarian organoid according to claim 92, wherein the ovarian germ cells are human germ cells.

94. The ovarian organoid according to claim 92 or 93, wherein the ovarian germ cells are primary germ cells (PGCs) or primary germ cell-like cells (PGCLCs).

95. The ovarian organoid according to claim 94, wherein the primary germ cells (PGCs) or primary germ cell-like cells (PGCLCs) undergo meiotic transition.

96. The ovarian organoid according to any one of claims 92 to 95, wherein the ovarian germ cells differentiate within the ovarian organoid, and the differentiated ovarian germ cells have a phenotype similar to that of human fetal germ cells derived from in vivo tissue.

97. The ovarian organoid according to any one of claims 92 to 96, wherein the differentiated ovarian germ cells express one or more primordial germ markers, one or more oogonia markers, one or more markers for meiotic initiation, and / or one or more oocyte markers.

98. The ovarian organoid according to any one of claims 92 to 97, wherein the differentiated ovarian germ cells express germ cell stage markers of human fetal germ cells derived from in vivo tissue.

99. The ovarian organoid according to any one of claims 92 to 98, wherein the ovarian germ cells differentiate within the ovarian organoid, and the differentiated ovarian germ cells have a phenotype similar to that of the germ cell stage of human fetal germ cells derived from in vivo tissue.

100. The ovarian organoid according to claim 99, wherein the germ cell stages progress from primordial germ cells, oogonia, meiosis initiation, and oocytes.

101. The ovarian organoid according to any one of claims 92 to 100, wherein the percentage of oocytes, oogonia, and / or other germ cells in the ovarian organoid is greater than 1%, greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, or greater than 50% of the total number of cells in the ovarian organoid.

102. The ovarian organoid according to any one of claims 92 to 101, wherein the ovarian germ cells of the cell mixture exhibit viability of at least 1%, at least 5%, at least 10%, at least 20%, at least 50%, or more after at least one week in culture.

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